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Peptidomics Methods and Protocols 1st Edition Mikhail
Soloviev (Auth.) Digital Instant Download
Author(s): Mikhail Soloviev(auth.), Mikhail Soloviev(eds.)
ISBN(s): 9781607615347, 1607615347
Edition: 1
File Details: PDF, 8.00 MB
Year: 2010
Language: english

METHODS IN MOLECULAR BIOLOGY
TM
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to
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Peptidomics
Methods and Protocols
Edited by
Mikhail Soloviev
RoyalHolloway,University ofLondon,Egham,UK

Editor
Mikhail Soloviev
Royal Holloway University of
London
School of Biological Sciences
Egham Hill
Egham, Surrey
United Kingdom TW20 0EX
mikhail.soloviev@rhul.ac.uk
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-60761-534-7 e-ISBN 978-1-60761-535-4
DOI 10.1007/978-1-60761-535-4
Library of Congress Control Number: 2009940608
© Humana Press, a part of Springer Science+Business Media, LLC 2010
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Preface
Despite being known and studied for years, peptides have never before attracted enough
attention to necessitate the invention of the term “Peptidomics” in order to specify the
study of the complement of peptides from a cell, organelle, tissue or organism. This vol-
ume presents a comprehensive range of analytical techniques for analysis of the peptide
contents of complex biological samples. The emphasis is often on higher throughput
techniques, suitable for the analysis of large numbers of peptides typically present in the
peptidomes or other complex biological samples. A wide range of methods is presented,
covering all stages of peptidomic research including, where applicable, organism handling,
tissue and organ dissection, cellular and subcellular fractionation, peptide extraction, frac-
tionation and purification, structural characterisation, molecular cloning and sequence
analysis. In addition to this, a selection of methods suitable for quantification, display,
immunochemical and functional analysis of peptides and proteins are presented. The
methods and techniques covered in this volume encompass a number of species rang-
ing from bacteria to man and include model organisms such as Caenorhabditis elegans,
Drosophila melanogaster and Mus musculus. Strong emphasis is placed on data analysis,
including mass spectra interpretation and in silico peptide prediction algorithms. Where
relevant, the peptidomic approaches are compared to the proteomic methods. Here is a
snapshot of the practical information, peptidomic methods and other related protocols
included in this volume:
Target organisms and samples covered: Bacteria (Chapter 2), hydra (Chapter 21), nema-
tode (Chapter 3), mollusc (Chapter 4), crab (Chapter 5), spider venoms (Chapters 6
and 7), insects (Chapters 8, 9, 10, 11, 25), amphibians (Chapters 12, 13, 14), rodents
(Chapters 15, 16, 17, 18), samples of human origin (Chapters 19, 20, 22, 23) and plants
(Chapter 26).
Peptide extraction and Liquid chromatography fractionation methods (mostly size
exclusion, ion exchange, reverse-phase modes or their combinations) can be found in
Chapters 2, 3, 4, 5, 6, 7, 8, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22). These include OFF-
line and ON-line techniques. The former are often used with MALDI-MS detection (e.g.
Chapters 3, 4, 5, 6, 7, 8, 12, 15, 16, 19) whilst the latter more generally with single or
multidimensional hyphenated LCN
-MSN
techniques (e.g. Chapters 2, 3, 15, 17, 18, 21).
Other separation and fractionation methods covered include microdialysis of live ani-
mals (Chapter 5), SDS-PAGE (Chapters 6, 18), magnetic bead based purification (Chap-
ter 20) and solid-phase extraction (Chapters 2, 6, 12, 19, 22).
Affinity peptide detection including anti-peptide antibody development and characteri-
sation, Affinity peptidomics, ELISA and microarray affinity assays are covered in Chapters
22, 23 and 24.
Mass spectrometry techniques include MALDI-TOF MS (e.g. Chapters 3, 6, 7, 8, 12,
16, 19, 20), MALDI-TOF with PSD (Chapter 8), MALDI-TOF MS/MS (e.g. Chapters
4, 6, 15, 21); ESI-MS/MS techniques (Chapters 3, 6, 16, 17, 18) or high-resolution
FTMS (Chapter 2). Direct MALDI-MS peptide profiling from cells and tissues is described
in Chapters 9, 10 and 11.
v

vi Preface
The description of functional assays can be found in Chapters 7, 14 and 21. Of par-
ticular interest in this respect is Chapter 21, where functional activity of the peptides is
assessed through the analysis of mRNA transcription levels changes in response to the
peptide application. That chapter contains a selection of protocols for peptide extraction,
fractionation and functional testing using a combination of molecular biology techniques,
cellular and morphological assays.
Molecular cloning of peptide cDNAs and the associated techniques are described in
Chapters 13 and 14.
Issues related to peptide sequence analysis are addressed in many chapters dealing with
MS spectra interpretation, but of special interest in this respect are Chapters 25 and 26,
dealing with in silico peptide prediction techniques and Chapter 20 which includes a section
on bioinformatics analysis of peptide expression profiling data. Differential peptide expression
issues are also covered in Chapter 2.
Peptidomics is 10-years old. My congratulations go to all scientists who have created
and developed the science of Peptidomics through their research and especially those who
found time to contribute their invaluable know-how in the form of methods and protocols
for inclusion in this volume. Peptidomics: Methods and Protocols is designed to complement
previously published titles in the Methods in Molecular BiologyTM
series, which focused on
protein analysis. This volume will help the beginner to become familiar with this fascinating
field of research and will provide scientists at all levels of expertise with easy-to-follow
practical advice needed to set up and carry out analysis of the peptide contents of complex
biological samples.
Royal Holloway University of London Mikhail Soloviev
December 2009

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
SECTION I INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1. Peptidomics: Divide et Impera . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Mikhail Soloviev
SECTION II FROM BACTERIA TO MEN . . . . . . . . . . . . . . . . . . . . . . . . 11
2. Performing Comparative Peptidomics Analyses
of Salmonella from Different Growth Conditions . . . . . . . . . . . . . . . . . 13
Joshua N. Adkins, Heather Mottaz, Thomas O. Metz, Charles Ansong,
Nathan P. Manes, Richard D. Smith, and Fred Heffron
3. Approaches to Identify Endogenous Peptides in the Soil Nematode
Caenorhabditis elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Steven J. Husson, Elke Clynen, Kurt Boonen, Tom Janssen,
Marleen Lindemans, Geert Baggerman, and Liliane Schoofs
4. Mass Spectrometric Analysis of Molluscan Neuropeptides . . . . . . . . . . . . 49
Ka Wan Li and August B. Smit
5. Monitoring Neuropeptides In Vivo via Microdialysis
and Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Heidi L. Behrens and Lingjun Li
6. Protocols for Peptidomic Analysis of Spider Venoms . . . . . . . . . . . . . . . 75
Liang Songping
7. Purification and Characterization of Biologically Active Peptides
from Spider Venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Alexander A. Vassilevski, Sergey A. Kozlov, Tsezi A. Egorov,
and Eugene V. Grishin
8. MALDI-TOF Mass Spectrometry Approaches
to the Characterisation of Insect Neuropeptides . . . . . . . . . . . . . . . . . 101
Robert J. Weaver and Neil Audsley
9. Direct MALDI-TOF Mass Spectrometric Peptide Profiling
of Neuroendocrine Tissue of Drosophila . . . . . . . . . . . . . . . . . . . . . 117
Christian Wegener, Susanne Neupert, and Reinhard Predel
10. Direct Peptide Profiling of Brain Tissue by MALDI-TOF
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Joachim Schachtner, Christian Wegener, Susanne Neupert,
and Reinhard Predel
vii

viii Contents
11. Peptidomic Analysis of Single Identified Neurons . . . . . . . . . . . . . . . . . 137
Susanne Neupert and Reinhard Predel
12. Identification and Analysis of Bioactive Peptides
in Amphibian Skin Secretions . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
J. Michael Conlon and Jérôme Leprince
13. An Efficient Protocol for DNA Amplification of Multiple Amphibian
Skin Antimicrobial Peptide cDNAs . . . . . . . . . . . . . . . . . . . . . . . . 159
Shawichi Iwamuro and Tetsuya Kobayashi
14. Combined Peptidomics and Genomics Approach to the Isolation
of Amphibian Antimicrobial Peptides . . . . . . . . . . . . . . . . . . . . . . . 177
Ren Lai
15. Identification and Relative Quantification of Neuropeptides
from the Endocrine Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Kurt Boonen, Steven J. Husson, Bart Landuyt, Geert Baggerman,
Eisuke Hayakawa, Walter H.M.L. Luyten, and Liliane Schoofs
16. Peptidome Analysis of Mouse Liver Tissue by Size Exclusion
Chromatography Prefractionation . . . . . . . . . . . . . . . . . . . . . . . . . 207
Lianghai Hu, Mingliang Ye, and Hanfa Zou
17. Rat Brain Neuropeptidomics: Tissue Collection, Protease Inhibition,
Neuropeptide Extraction, and Mass Spectrometric Analysis . . . . . . . . . . . . 217
Robert M. Sturm, James A. Dowell, and Lingjun Li
18. Quantitative Neuroproteomics of the Synapse . . . . . . . . . . . . . . . . . . 227
Dinah Lee Ramos-Ortolaza, Ittai Bushlin, Noura Abul-Husn,
Suresh P. Annangudi, Jonathan Sweedler, and Lakshmi A. Devi
19. Peptidomics Analysis of Lymphoblastoid Cell Lines . . . . . . . . . . . . . . . 247
Anne Fogli and Philippe Bulet
20. Peptidomics: Identification of Pathogenic and Marker Peptides . . . . . . . . . . 259
Yang Xiang, Manae S. Kurokawa, Mie Kanke, Yukiko Takakuwa,
and Tomohiro Kato
SECTION III TOOLS AND APPROACHES . . . . . . . . . . . . . . . . . . . . . . . . 273
21. Peptidomic Approaches to the Identification and Characterization
of Functional Peptides in Hydra . . . . . . . . . . . . . . . . . . . . . . . . . 275
Toshio Takahashi and Toshitaka Fujisawa
22. Immunochemical Methods for the Peptidomic Analysis
of Tachykinin Peptides and Their Precursors . . . . . . . . . . . . . . . . . . . 293
Nigel M. Page and Nicola J. Weston-Bell
23. Affinity Peptidomics: Peptide Selection and Affinity Capture
on Hydrogels and Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Fan Zhang, Anna Dulneva, Julian Bailes, and Mikhail Soloviev

Contents ix
24. In Situ Biosynthesis of Peptide Arrays . . . . . . . . . . . . . . . . . . . . . . . 345
Mingyue He and Oda Stoevesandt
25. Bioinformatic Approaches to the Identification of Novel Neuropeptide
Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Elke Clynen, Feng Liu, Steven J. Husson, Bart Landuyt,
Eisuke Hayakawa, Geert Baggerman, Geert Wets, and Liliane Schoofs
26. Bioinformatic Identification of Plant Peptides . . . . . . . . . . . . . . . . . . . 375
Kevin A. Lease and John C. Walker
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385

Contributors
NOURA ABUL-HUSN • Department of Pharmacology and Systems Therapeutics, Mount
Sinai School of Medicine, New York, NY, USA
JOSHUA N. ADKINS • Fundamental and Computational Sciences Directorate, Pacific
Northwest National Laboratory, Richland, WA, USA
SURESH P. ANNAGUDI • Department of Chemistry and the Beckman Institute,
University of Illinois at Urbana-Champaign, Urbana, IL, USA
CHARLES ANSONG • Fundamental and Computational Sciences Directorate, Pacific
NEIL AUDSLEY • The Food and Environment Research Agency, Sand Hutton, York, UK
GEERT BAGGERMAN • ProMeta, Interfacultary Center for Proteomics and
Metabolomics, K.U. Leuven, Leuven, Belgium
JULIAN BAILES • School of Biological Sciences, Royal Holloway University of London,
Egham, Surrey, UK
HEIDI L. BEHRENS • Department of Chemistry, University of Wisconsin-Madison,
Madison, WI, USA
KURT BOONEN • Functional Genomics and Proteomics Research Unit, Department of
Biology, K.U. Leuven, Leuven, Belgium
PHILIPPE BULET • TIMC-IMAG, UMR 5525, Domaine de Chosal, Archamps, France
ITTAI BUSHLIN • Department of Pharmacology and Systems Therapeutics, Mount Sinai
School of Medicine, New York, NY, USA
ELKE CLYNEN • Functional Genomics and Proteomics, Department of Biology, K.U.
Leuven, Leuven, Belgium
J. MICHAEL CONLON • Department of Biochemistry, Faculty of Medicine and Health
Sciences, United Arab Emirates University, Al-Ain, UAE
LAKSHMI A. DEVI • Department of Pharmacology and Systems Therapeutics, Mount
Sinai School of Medicine, New York, NY, USA
JAMES A. DOWELL • Department of Chemistry, School of Pharmacy, University of
Wisconsin-Madison, Madison, WI, USA
ANNA DULNEVA • School of Biological Sciences, Royal Holloway University of London,
Egham, Surrey, UK
TSEZI A. EGOROV • Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian
Academy of Sciences, Moscow, Russia
xi

xii Contributors
ANNE FOGLI • GreD UMR INSERM 931 CNRS 6142, Faculté de Médecine,
Clermont-Ferrand, France
TOSHITAKA FUJISAWA • Institute of Zoology, University of Heidelberg, Heidelberg,
Germany
EUGENE V. GRISHIN • Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Moscow, Russia
EISUKE HAYAKAWA • Functional Genomics and Proteomics Research Unit, Department
of Biology, K.U. Leuven, Leuven, Belgium
MINGYUE HE • The Babraham Institute, Cambridge, UK
FRED HEFFRON • Department of Molecular Microbiology and Immunology, Oregon
Health and Sciences University, Portland, OR, USA
LIANGHAI HU • Key Laboratory of Separation Science for Analytical Chemistry,
National Chromatographic R&A Centre, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Dalian, China
STEVEN J. HUSSON • Functional Genomics and Proteomics, Department of Biology,
K.U. Leuven, Leuven, Belgium
SHAWICHI IWAMURO • Department of Biology, Faculty of Science, Toho University,
Funabashi, Chiba, Japan
TOM JANSSEN • Functional Genomics and Proteomics, Department of Biology, K.U.
MIE KANKE • Clinical Proteomics and Molecular Medicine, St. Marianna University
Graduate School of Medicine, Kawasaki, Japan
TOMOHIRO KATO • Clinical Proteomics and Molecular Medicine, St. Marianna
University Graduate School of Medicine, Kawasaki, Japan
TETSUYA KOBAYASHI • Department of Regulation Biology, Faculty of Science, Saitama
University, Saitama, Japan
SERGEY A. KOZLOV • Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Moscow, Russia
MANAE S. KUROKAWA • Clinical Proteomics and Molecular Medicine, St. Marianna
REN LAI • Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming,
Yunnan, China
BART LANDUYT • Functional Genomics and Proteomics Research Unit, Department of
Biology, K.U. Leuven, Leuven, Belgium
KEVIN A. LEASE • Division of Biological Sciences and Bond Life Sciences Centre,
University of Missouri, Columbia, MO, USA
JÉRÔME LEPRINCE • European Institute for Peptide Research (IFRMP 23), INSERM
U-413, UA CNRS, University of Rouen, Mont-Saint-Aignan, France

Contributors xiii
KA WAN LI • Department of Molecular and Cellular Neurobiology, Centre for
Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, VU
University Amsterdam, Amsterdam, The Netherlands
LINGJUN LI • Department of Chemistry, School of Pharmacy, University of
MARLEEN LINDEMANS • Functional Genomics and Proteomics, Department of Biology,
K.U. Leuven, Leuven, Belgium
FENG LIU • Data Analysis and Modeling Group, Transportation Research Institute,
Hasselt University, Diepenbeek, Belgium
WALTER H.M.L. LUYTEN • Department Woman and Child, Faculty of Medicine, K.U.
NATHAN P. MANES • Fundamental and Computational Sciences Directorate, Pacific
THOMAS O. METZ • Fundamental and Computational Sciences Directorate, Pacific
HEATHER MOTTAZ • Fundamental and Computational Sciences Directorate, Pacific
SUSANNE NEUPERT • Institute of General Zoology and Animal Physiology,
Friedrich-Schiller-University, Jena, Germany
NIGEL M. PAGE • School of Life Sciences, Kingston University London,
Kingston-upon-Thames, Surrey, UK
REINHARD PREDEL • Institute of General Zoology and Animal Physiology,
Friedrich-Schiller-University, Jena, Germany
DINAH LEE RAMOS-ORTOLAZA • Department of Pharmacology and Systems
Therapeutics, Mount Sinai School of Medicine, New York, NY, USA
JOACHIM SCHACHTNER • Department of Biology, Animal Physiology,
Philipps-University, Marburg, Germany
LILIANE SCHOOFS • Functional Genomics and Proteomics Research Unit, Department
of Biology, K.U. Leuven, Leuven, Belgium
AUGUST B. SMIT • Department of Molecular and Cellular Neurobiology, Centre for
Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, VU
University Amsterdam, Amsterdam, The Netherlands
RICHARD D. SMITH • Fundamental and Computational Sciences Directorate, Pacific
MIKHAIL SOLOVIEV • School of Biological Sciences, Royal Holloway University of
London, Egham, Surrey, UK
LIANG SONGPING • College of Life Sciences, Hunan Normal University, Changsha,
China
ODA STOEVESANDT • Babraham Bioscience Technologies, Cambridge, UK

xiv Contributors
ROBERT M. STURM • Department of Chemistry, School of Pharmacy, University of
JONATHAN SWEEDLER • Department of Chemistry and the Beckman Institute,
University of Illinois at Urbana-Champaign, Urbana, IL, USA
TOSHIO TAKAHASHI • Suntory Institute for Bioorganic Research, Osaka, Japan
YUKIKO TAKAKUWA • Clinical Proteomics and Molecular Medicine, St. Marianna
ALEXANDER A. VASSILEVSKI • Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry, Russian Academy of Sciences, Moscow, Russia
JOHN C. WALKER • Division of Biological Sciences and Bond Life Sciences Center,
University of Missouri, Columbia, MO, USA
ROBERT J. WEAVER • The Food and Environment Research Agency, Sand Hutton, York,
UK
CHRISTIAN WEGENER • Emmy Noether Neuropeptide Group, Animal Physiology,
Philipps-University, Marburg, Germany
NICOLA J. WESTON-BELL • Genetic Vaccine Group, Cancer Sciences Division,
Southampton General Hospital, University of Southampton School of Medicine,
Southampton, Hampshire, UK
GEERT WETS • Data Analysis and Modeling Group, Transportation Research Institute,
Hasselt University, Diepenbeek, Belgium
YANG XIANG • Clinical Proteomics and Molecular Medicine, St. Marianna University
Graduate School of Medicine, Kawasaki, Japan
MINGLIANG YE • Key Laboratory of Separation Science for Analytical Chemistry,
National Chromatographic R&A Centre, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Dalian, China
FAN ZHANG • School of Biological Sciences, Royal Holloway University of London, Egham,
Surrey, UK
HANFA ZOU • Key Laboratory of Separation Science for Analytical Chemistry, National
Chromatographic R&A Centre, Dalian Institute of Chemical Physics, Chinese Academy
of Sciences, Dalian, China

Chapter 1
Peptidomics: Divide et Impera
Mikhail Soloviev
Abstract
The term “peptidomics” can be defined as the systematic analysis of the peptide content within a cell,
organelle, tissue or organism. The science of peptidomics usually refers to the studies of naturally occur-
ring peptides. Another meaning refers to the peptidomics approach to protein analysis. An ancient Roman
strategy divide et impera (divide and conquer) reflects the essence of peptidomics. Most effort in this field
is spent purifying and dividing the peptidomes, which consist of tens, hundreds or sometimes thousands
of functional peptides, followed by their structural and functional characterisation. This chapter intro-
duces the concept of peptidomics, outlines the range of methodologies employed and describes key
targets – the peptide groups which are often sought after in such studies.
Key words: Peptidomic, peptidome, peptide, functional peptide, methods.
1. Introduction
Polypeptides, being short stretches of amino acids or small pro-
teins, occupy a strategic position between proteins and amino
acids and play, for the most part, fundamental roles by regulat-
ing the vast majority of biological processes in the animal king-
dom. Whilst perhaps sometimes overlooked, the importance of
the regulatory role of peptides is truly great and hard to overes-
timate. Peptides have in fact been the focus of much research for
decades with the first successful attempts to analyse peptide con-
tent of various biological samples (including from urine, blood
and brain tissues) having been reported over 60 years ago. These
relied mostly on chromatography, including two-dimensional liq-
uid chromatography (LC), or mass spectrometry (MS) tech-
niques. Recent advances in MS and further developments in liquid
chromatography, including nano-LC (1) and associated “omics”
M. Soloviev (ed.), Peptidomics, Methods in Molecular Biology 615, DOI 10.1007/978-1-60761-535-4 1,
© Humana Press, a part of Springer Science+Business Media, LLC 2010
3

4 Soloviev
techniques, resulted in dramatic improvements in the sensitivity
and high throughput of protein and peptide analyses (2) and gen-
erated unprecedented growth in the number of relevant publica-
tions (Fig. 1.1).
0.0%
0.1%
0.2%
0.3%
0.4%
0.5%
0.6%
0.7%
0.8%
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
0.000%
0.001%
0.002%
0.003%
0.004%
0.005%
0.006%
0.007%
0.008%
Peptidomics
Proteomics
Fig. 1.1. Peptidomics publications since 1999. The bars represent the number of publications in PubMed containing
“peptidomics OR peptidomic OR peptidome” normalised to the total number of publications added to PubMed each year.
Proteomics publications in PubMed (found similarly) follow the same trend (solid line). Vertical axes show normalised
data (in %) for proteomics papers (left) and peptidomics papers (right). Total number of publications on 1 January 2009
was 28,273 (proteomics) and 246 (peptidomics).
2. Peptidomics
of Naturally
Occurring
Peptides and
Peptide Pools
Similarly to “proteomics”, the term “peptidomics” can be defined
as the systematic analysis of the peptide content within an organ-
ism, tissue or cell (3) in order to determine peptides’ identity,
quantity, structure and function. Such interpretation made its
public debut at the 2nd International Seminar on the Enabling
Role of MS in 1999 (4), before finally appearing in press in 2001
in research papers by Peter D.E.M. Verhaert et al. (5), Peter
Schulz-Knappe et al. (6) and Elke Clynen et al. (7). The disci-
pline of peptidomics focuses on peptides that often display bio-
logical activity such as hormones, cytokines, toxins, neuropeptides
and alike, which are generated from larger precursors, as well as
biomarker-type peptides that may not have any bioactivity but are
indicative of a particular pathology, for example the up/down-
regulation of many serum peptides that result from proteolysis.

Peptidomics: Divide et Impera 5
Peptidomics is in its infancy relative to other “omics” (28,273
papers on PubMed for a “proteomics” search compared to just
246 for “peptidomics” as of January 1st, 2009) but is expanding
rapidly (Fig. 1.1).
3. Peptidomics
Approach to
Proteomics
In parallel, and independently of the peptidomics definition given
in (5–7), another meaning was introduced by Barry et al. (8)
in relation to the analysis of peptide pools (of biological fluids,
tissues or cells) obtained by means of proteolytic digestion of
these samples and in particular using affinity-based analysis (hence
“Affinity peptidomics”) e.g. in the form of protein arrays (9, 10).
Since biologically occurring peptides (whether biologically active
or not) are strictly speaking also the products of proteolysis (e.g.
insulin pre-pro-insulin, or biologically active peptides obtained
through “non-specific” proteolysis of e.g. haemoglobin), both
definitions of “peptidomics” are therefore very similar in that they
refer to the analysis of partially or fully proteolytically digested
proteins, i.e. peptides. And finally, to acknowledge everyone
involved in the birth of the “peptidomics” as a separate field of
chemical biology, we should mention a Germany-based company
“BioVisioN AG” which filed a trademark “peptidomics” in 1999
to cover “Chemicals used in science, in particular for analysis,
other than for medical or veterinary purposes”; “Medical, veteri-
nary and pharmaceutical products” as well as the “Scientific and
industrial research; conducting medical and non-medical analyses;
services in the field of diagnostics; development of pharmaceutical
active substances; purchasing, licensing and exploitation of intel-
lectual property” (11).
4. Peptidomics:
The
Methodologies
In the past, the majority of separation techniques used in pro-
tein and peptide analysis relied on their physical properties, such
as protein or peptide size, shape, polarity, pI, the distribution of
ionisable, polar and non-polar groups on the molecule surface,
and their affinity towards specific or non-specific affinity capture
reagents. Modern separation techniques rely on a combination of
isoelectric focusing, electrophoretic separation and a great vari-
ety of liquid chromatography techniques, often linked together
to yield two- or three-dimensional separation approaches, and

6 Soloviev
frequently backed up by serious automation. Highly parallel anal-
ysis is often attempted through miniaturisation (12) and the use
of chip-based techniques (13–16) or the Agilent 2100 Bioan-
alyzer (www.chem.agilent.com). The inherent heterogeneity of
the proteins’ and to a lesser degree peptides’ physical properties
which underlies all of the above separation options is, at the same
time, the inherent problem of any highly parallel protein analy-
sis. A single universal system suitable for extraction and separa-
tion (let alone functional analysis) of all classes of proteins is yet
to be reported. Unlike proteins, the peptides are often less het-
erogeneous in their physico-chemical properties and therefore the
complete peptidomic analysis of samples, tissues and in some cases
whole organisms is more straightforward than proteomic analysis.
In addition to physical methods of analysis and separation,
chemical biology offers a number of other approaches, which rely
directly or indirectly on chemical modifications and separation
principles based on chemical properties of proteins and peptides.
Chemical modification of the side chains of proteins and peptides
was first reported many decades ago (see 17 for a review) and
has been used widely since for protein modifications, labelling
and cross-linking, but not so widely for protein separations – the
latter because of the issues related to the availability and surface
exposure of the reactive groups. Unlike proteins, peptides offer
a unique chance to apply chemical selection techniques because
of the lack of complex secondary structure and virtually com-
plete exposure to solvent of all of the reactive groups. A number
of reports utilising chemical biology approach to peptide sepa-
ration and analysis have been published more recently. In most
cases these describe various group-specific labelling procedures,
often linked to peptide quantification (18, 19) as well as chemical
depletion approaches (17, 20). Among the other “omics” tech-
nologies and approaches, “peptidomics” is the most comparable
to “Proteomics” and although the terms are not synonymous, the
underlying techniques and approaches are almost identical. For
example, MS, a cornerstone of modern proteomics, in most cases
actually analyses peptides (obtained through proteolytic digestion
of proteins) or their fragments (obtained through e.g. CID), not
proteins. The difference between the terms “peptidomics” and
the “proteomics” is therefore blurred, especially if “methods” are
being considered.
5. Peptidomics:
The Targets
Target-wise, the peptidomics research is often focused, although
not always, on studying peptides formed in vivo by proteolysis of
specialised or non-specialised precursor proteins (often bioactive

Peptidomics: Divide et Impera 7
peptides), rather than “artificially” or in vitro-produced peptides.
The range of biological activities displayed by naturally occur-
ring peptides is truly remarkable; it ranges from toxins that can
paralyse or kill to peptides that have the ability to heal. The ven-
oms of arthropods such as spiders and scorpions, as well as other
species such as cone snails, comprise a vast number of neuromod-
ulatory peptides that are capable of serious harm, but also serve
as a highly useful point to discover new drugs such as painkillers
(21). The identification and functional characterisation of pep-
tides from all species including humans is crucial in the discovery
of novel biomarkers and drug targets, and may yield novel ther-
apeutic agents such as peptide-based vaccine Glatiramer acetate
(GA) (Copaxone) used for the treatment of relapsing and remit-
ting cases of multiple sclerosis (22). The suitability of peptides
as biomarkers stems from the fact that they are present in all
body fluids, cells and tissues (23), and many approaches focus
on identifying them from such samples (24, 25). Peptides also
play crucial roles in innate and adaptive immune responses by
forming complexes with MHC-I, MHC-II and T cells where they
stimulate defensive immune responses (26–28). The importance
of peptides in cell-to-cell communication underpins the impor-
tance of peptidomics in understanding multiple pathologies that
result from these communication processes going wrong. The
peptide content of biological fluids, such as urine for example,
can be used to produce a complete peptidomic fingerprint of
an individual’s health (29). The evolutionary evidence to sup-
port the importance of peptides in such widespread biological
roles is evident when one examines the conservation of peptide
families across species. The Tachykinin peptides for example, the
largest known neuropeptide family, are found in vertebrates, pro-
tochordates and invertebrates (30). On the other end of the scale,
even primitive microorganisms rely on peptide signalling, such as
for example bacterial quorum sensing (31, 32) and yeast mating
factors (33).
The following chapters provide a comprehensive guide to
peptidomics methods and applications, spanning a range of
species from bacteria to man and covering a wide range of relevant
methods from basic biochemistry techniques to in silico tools and
protocols.
References
1. Chervet, J.P., Ursem, M., and Salzmann,
J.B. (1996) Instrumental requirements for
nanoscale liquid chromatography. Anal.
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2. Quadroni, M. and James, P. (1999) Pro-
teomics and automation. Electrophoresis 20,
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3. Schrader, M. and Schulz-Knappe, P.
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and Rossier, J.S. (2004) Why the move
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Forssmann, W.G. (2004) Exploiting natu-
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different content

yet, as he considered their position, he realized its dangers and soon
lapsed into silence.
Trusting to the cover of night, the quartette rode along the highway
which made a swing toward the health resort that had proved
anything but healthy for them, as by doing so they could strike a
road that would connect more directly with one running in the
direction of Ste. Genevieve.
For now that he had made up his mind to loot his enemy's bank, he
determined to travel by the most direct route to the town in which it
was located. Yet before they arrived, it would be necessary for them
to cross the State of Missouri, Ste. Genevieve being situated on the
shore of the Mississippi river.
"It'll throw a shock into 'em to hear that we've raided old Rozier's
shebang when they think we're hiding somewhere near the Springs,"
chuckled Jesse, all of a sudden.
"That it will," echoed Cole almost in the same breath and the latter
continued: "They say it's a rich bank, too. Always has a hundred
thousand or so in the vaults."
"So I understand," rejoined the great outlaw. "But it'll never have so
much again. When we get through with it, people will be afraid to
put their money in it. I mean to leave word that I shall raid it every
year!
"Before I get through with that cur, Rozier, he'll be sorry he ever
insulted me and then set detectives on my trail!"
The bitterness with which he uttered the words and the threat told
how deeply the bank president's actions had cut the world-famous
desperado. The disclosure so amazed his hearers that they were
unable to make adequate comment and ere any occurred to them,
their attention was distracted by the shrill neigh of a horse in front of
them.

Not more than four miles from the Springs were the bandits when
the startling sound broke on the air.
Visions of patrols and posses instantly flashed to their minds and
they drew rein hurriedly.
"Into the bushes, lively! Everybody on the same side," snapped
Jesse. "We'll just see who's riding so late at night."
But the desperadoes were entirely unprepared for the sight that met
their eyes a few minutes after they had hidden themselves.
The spot Jesse had chosen from which to spy on the travellers was
his favorite location for an ambuscade, a turn in the road.
As the dull pounding of the hoofs came nearer and nearer, the
bandits peered eagerly from the protecting bushes, pistols in their
hands for immediate use should their chief give the word.
But as they gazed at the highway, its white course seeming like
some huge piece of ribbon stretched along the ground, they were
amazed to see a girl and a man sweep around the bend, followed
close by two more women.
Each of the three females was heavily veiled and wrapped in shawls
that covered the saddles.
More than that the desperadoes could not see for the late riders
were going at a fast gallop.
"Probably been to some party," commented Jim when they had
disappeared up the road.
"They're mighty quiet if they have," declared John.
"Righto! They act more to me like people who are trying to escape
from some one," interposed Jesse.
"Maybe they've heard we were in the neighbourhood and are
keeping quiet so they won't attract us," suggested Cole.

"Non—" began his chief, then suddenly stopped, whistled low and
gasped:
"By thunder! They may have been Sue and some of the Priors.
Perhaps the people have made it so hot for them they've had to flee.
Come on, we'll follow and find out!
"By the blood of my mother! If the citizens of the Springs have
driven them out, we'll go back and shoot that burg up if we never do
another thing!"
Yet before they could put the words into action they were treated to
a second, and still greater, surprise.
The sound of fast running horses again fell on their ears from the
direction whence the three women and man had come.
"Sit tight," whispered Jesse. "There's something in the wind. We'll
follow when they get by."
Scarcely had the words left his lips than around the turn in the
highway dashed six horsemen, rifles at a ready.
Fearful lest they hear the crackling of the bushes, the bandits waited
till the hoof-beats were almost inaudible, then bounded into the
roadway and gave chase.
Riding like fiends till they were within sight of the last group of
equestrians, Jesse slowed down to a pace at which he could watch
them from a safe distance.
For minutes, that seemed hours, the strange procession advanced,
the first group in ignorance of the two behind it and the second
unaware that it was followed.
Then, of a sudden, a voice from a horseman in the middle shouted:
"Halt, or we'll shoot!"

Rising in their stirrups the better to see, the desperadoes awaited
the result of the command.
But instead of obeying, those in the lead only increased their speed.
There was a moment of intense silence.
Then a rifle cracked.
And before the report died away a woman's scream rang out.
"By the blood of my mother! That's Sue's voice!" gasped the world-
famous outlaw. "After the devils, boys! We must get 'em before they
can shoot again!
"If they've hurt my sister or the girls with her, their life-blood will pay
for it!"
Roused by the cowardly shot at the helpless women, the Younger
brothers sprang their horses after their leader, ranging themselves at
his side when they had, with difficulty, overtaken him.
Having been prevented from shooting at their pursuers when they
made their dash for liberty from the Prior farm by their lack of rifles,
Jesse had lost no time in supplying the want when he had
discovered the small arsenal possessed by Theodoric Snuffer, though
his eagerness to purchase them compelled him to pay an
exhorbitant price, and as they cut down the distance between the
six horsemen and themselves, he ordered his companions to unsling
the Winchesters from their shoulders.
The solitary shot, however, had been sufficient to cause the girls and
their escort to draw rein.
"We've got to get 'em before they reach Sue and the others or we
may hit them instead," hissed the great outlaw. "Rise in your
stirrups! Ready; Fire!"
As the crash of the volley broke on the air, the six horsemen whirled
in their saddles.

But before they could see whence the unexpected sound came, two
of them toppled from their saddles while the horse of another fell in
the roadway.
"Again!" yelled Jesse.
Yet in response to the second discharge of their rifles only one man
fell.
"Steady, boys! Aim higher!" cautioned their chief. "One more volley
and then we'll rush 'em!"
Recovering from the surprise of the attack, the remaining members
of the middle group blazed away with their Winchesters.
"Out to the edge of the road!" commanded the bandit-chieftain, his
voice sounding terrible in its fury.
Instantly his companions obeyed while the bullets whistled about
them.
Though the move forced them to advance at a slower gait, it was a
wise one, for their figures no longer loomed from the white roadbed,
leaving their foes at a loss where to aim their rifles.
Incessantly the bandits pumped their rifles till only one of the six
remained.
"Surrender, or we'll kill you!" shouted the world-famous desperado.
Unnerved by the slaughter of his comrades, the survivor gasped:
"All right. I do."
"Then drop your gun and throw up your hands," thundered the great
outlaw and as the man obeyed, he burst into a laugh hideous in its
uncanny hollowness.
Like a whirlwind, the bandits bore down upon the cowering creature.

"Keep him covered!" snapped the terrible desperado to his pals, then
raising his voice yelled: "Don't be afraid Sue. It's only I."
As she heard her name, the girl scarcely believed her ears, then,
urging her horse forward, she rode to meet her brother, crying:
"Oh, Jess! Is it really you!"
"Surest thing you know," returned the world-famous bandit. "Are
you hurt, girlie? I heard your scream. That's how I recognized you."
"Not a scratch. It was the shock, I guess. It was so unexpected."
And unable to control her overwrought nerves she burst into tears.
"There, there, sis, don't cry. It's all right, so long as you're not hurt,"
soothed her brother, taking the trembling girl in his arms. "But it's a
lucky thing. I happened to be in this vicinity."
Taking out his flask, Jesse ordered his sister to drink some of the
brandy.
"It won't do any of the rest of you any harm to swallow some," he
declared, turning to his sister's companions. "Who are you,
anyhow?"
Forgetting her desire to faint in her eagerness to answer the
question herself, Susie exclaimed:
"They're Marjorie and Helen—"
"And Jeff," hazarded the bandit-chieftain.
"No—o."
"Who, then?" demanded the great desperado, his voice harsh and
stern.
"It's T—Tim."
"Tim Mason? What on earth's he doing here?"

"He—he came down to meet me," faltered his sister.
"Did your mother know he was coming down?"
But ere Susie could reply, Tim took the bull by the horns, exclaiming:
"She did not! But it's all right."
"Oh, is it? What would you think if your sis—"
"Wait till I finish," interrupted the fellow, hotly. "We came down here
to be married."
The statement made, Tim rushed on, regardless of consequences:
"We got tired of waiting for you to keep your promise of giving your
consent to the wedding. And as Susie needs some one to protect her
from the detectives who haunt the house because of your—"
"Take care," hissed Jesse. "I'm in no pleasant mood to-night." But as
he caught the look of appeal cast him by his sister, his anger
vanished and taking her head between his hands, he laughed: "So
that was the cause of your coming to the Springs. Phew! An
elopement!
"Jicky, but it must have been an awful shock to you when you saw
Cole.
"You stood by like a trump, though, when we were caught in the
house and I guess you are entitled to a reward.
"Come here, Tim. Give me your hand—that's it. Now take Sue's.
There you are. You may have her, boy, but if I ever hear a word of
complaint of your treatment of her, well—you'd better light out for
the tall timber before I can hit your trail."
The dramatic scene of the betrothal, a lonely spot in the country, the
starless canopy of heaven above, the white road against which was
silhouetted the figure of the prisoner, hands above his head, his
captors covering him with their rifles, the huddled bodies of the dead
men at his feet, with no sounds save the creaking of the saddle

leather and their own breathing to break the awesome silence of the
night, impressed the actors profoundly.
From one to another they looked, the expressions on their faces
proclaiming they were aware that but for the timely interference,
bloody and terrible as it was, of the dread bandits they might even
at that moment be lying in the places of the lifeless forms.
"Come, this is time for mirth, not tears," rallied Jesse. "Boys, pull out
your flasks. We'll drink to the health and happiness of the future
Mrs. and Mr. Timothy Mason."
The tension broken, everybody talked at once as the flasks were
passed from one to another.
Brought back to their normal selves, the great outlaw asked the
meaning of the flight from the Springs, to learn of the crowd's cry
for the capture of the girls, their escape with the aid of Lawyer
Perkins, their hiding in his house, his going to the station to meet
Susie's lover at her request, the return of the man-hunters and the
decision of Tim and the barrister that it would be best for the girls to
leave the town under cover of the night.
Apprised of these points, the bandit-chieftain turned his attention to
his captive.
Striding so close that their bodies almost touched, he rasped:
"Why did you follow the girls? How did you know they were riding?"
"W—we thought they were going to meet you. We were going to
make them lead us to you and—"
"So that's why you shot at them, knowing they were women?" burst
in the terrible bandit.
"We only did it to frighten 'em."
"You certainly succeeded—and their screams brought death to your
fellows.

"Who were they?"
Quickly the fellow reeled off some names that Marjorie and Helen
declared belonged to none of whom they had ever heard.
"Who sent you, Dillaby or old Rozier?" thundered Jesse.
"Neither. We were going to join a posse to patrol the town when we
saw them ride from Lawyer Perkins' house."
"You're lying. I can tell by the sound of your voice," interrupted the
world-famous desperado. "But it doesn't make any difference.
"After we've taken your weapons away from you, I'm going to send
you back to the Springs.
"Now listen carefully. I want you to tell the people how you shot at
three defenceless women, how we surprised you and what we did to
your women-shooting companions.
"Tell them that if they ever try to harm a hair on the head of Jesse
James' sister, he'll hound them to hell!"
During the awful threat, Cole and his brothers had removed the
pistols, knives and cartridges from the prisoner's clothes and as the
famous outlaw saw they had finished the task, he roared:
"Now go, you cur! Ride for your life! If we can see you in two
minutes, we'll open fire at you! And don't forget to deliver my
message!"
Ere the last words had been uttered, however, the man, who
believed himself literally returned from the dead, whirled his horse
and dashed madly back toward the Springs.
And as he rode, the mocking laughter of Jesse rang in his ears.

Chapter XIV.
"PRINCE CHARMING."
"Can't we take a couple of shots at the guy?" implored John, casting
yearning glances in the direction of the furiously riding horseman.
But before his chief could reply, Susie exclaimed:
"No, please say no, Jesse. We've—ugh!—seen enough shooting
tonight," and she turned her large eyes, full of pleading, upon her
brother.
"As you say, sis," returned he, good-naturedly. "The ladies are in
command now, John, so I can't allow you to fire at the wretch,
though it does seem a shame not to give him a couple of bullets to
hurry him a bit."
Susie, however, was deaf to supplication and with a sigh the outlaws
turned their backs upon the lone rider.
"Are you going to leave those—er—men?" inquired Marjorie, as
Jesse and his companions dismounted to look to the cinches of their
saddles.
"We are," returned the bandit-chieftain with an emphasis that
warned the others that he had yielded to their demands as far as he
would. "They'll serve as a warning that my family and friends are not
to be trifled with."

Then dropping his harsh tone, he inquired: "Where were you-all
bound for when this interruption occurred?"
"To my aunt, Mrs. Jarvis, in Deepwater," answered Tim.
"Good. The girls ought certainly to be secure from annoyance with
any of your family, who'd never be suspected of harbouring the
Jameses or their friends. I rather think you girls had better stay
there for a few days till things get quieter. There's liable to be
something happen in course of forty-eight hours or so."
"Oh, Jess, why can't you be satisfied with getting away?" besought
his sister, only to be silenced by his retort:
"Just mind your own affairs, little girl. There are a good many things
you don't understand so don't bother your head about them. You'll
have enough to think of—and more—with silks and ribbons and all
the rest. For now that you and Tim have tried to steal a march on
me once, the sooner you are married the less likely you'll be to
attempt it again."
Their thoughts distracted from their nerve-racking experience
through which they had passed by the reference to the nuptials,
Susie and the girls, with women's interest in such events, fell to
discussing the clothes that would be necessary and, smiling
indulgently at their innocent prattle, the desperadoes ordered them
to advance and rode along in their rear.
In apparent forgetfulness of the presence of the men who were so
feared, Susie and her friends proceeded for miles before they
noticed that they were following.
"Why, Jess," exclaimed Marjorie, happening to look back over her
shoulder, "I didn't know you had come with us. Aren't we taking you
out of your way?"
"Never mind about that," rejoined the world-famous desperado. "It's
a good twenty miles from here to Deepwater and lots of things could
happen before you got there, so many that I don't propose to risk

your going alone. But if you want to do me a favour, will you ride
faster and keep quiet? There's no telling who's abroad."
Recalled to the reality of the danger they were running in traveling
at night when the whole section of the state was supposed to be
searching high and low for the dread Jesse and his companions, the
little troop rode on in silence.
As they approached a dark place in the highway the bandit-chieftain
and John galloped ahead to draw the fire, in case any patrols were
in ambush, while Cole and Jim guarded the rear. Villages and towns
were given a wide berth, the cavalcade making a wide detour
around them, even avoiding clusters of farm houses in their anxiety
not to arouse any dogs which might give the alarm and enable the
course of the fugitives to be traced.
At last, as the sky was growing green with the first tinge of the
coming day, the spires of Deepwater came in sight, their white sides
looking like spectres hovering between heaven and earth.
As they approached the town, the crowing of the cocks announced
the wakening life.
"I don't believe we'll go any farther," declared Jesse, calling a halt on
the outskirts of the village. "No one will interfere with you now and if
any body should, you can say you're guests of Mrs. Jarvis out for an
early ride. Tim knows how to work the bluff. I think you'd best get
back to Kearney this morning, boy. You can explain to mother about
Sue's trip to Monegaw Springs and tell her that it's all right for I've
given my consent. When you've eased her mind, you come back
here. Oh, you might tell her I'm going on a business trip to Ste.
Genevieve and that I'll run in to see her on my way back. Say that
Frank and Texas have gone north on a surveying expedition to be
gone several weeks. If she, or any of you, have anything important
to communicate to me, you can meet me in the old wood-chopper's
camp at Sni Mills in five weeks.
"Take care of yourselves, now."

And touching his lips to his sister's forehead, the dare-devil bandit
turned, caught Marjorie around the waist and planted a resounding
smack on her luscious mouth, repeated the salute on Helen and
cantered away, laughing gaily, before the blushing girls could
remonstrate.
The deviation from his course across the state, necessitated by the
escorting of his sister and friends to their destination, was little to
the taste of the world-famous desperado, though he concealed his
feelings from Susie and the girls.
But once clear of them, he rode like mad to recover the lost ground.
His hope of success in looting the bank lay in striking before the
man-hunters should discover that he had left the region of Monegaw
Springs and in his race against time, every hour was precious.
Keeping to the highway till it grew so light they feared discovery, the
outlaws finally rode into the woods and when they reached a well-
sheltered ravine, dismounted to rest their horses.
Though Cole and Jesse had left their false beards in the little ham-
chamber in the chimney of the Prior house, where they had suffered
such agony, they still wore their business suits they had donned in
McAlester, in the Indian Territory, before going to the Springs.
John and Jim, however, were clad in the cowboy garb the bandits
affected. "Wouldn't it be best for us to shift back into our 'Wild West'
togs?" suggested the eldest of the Younger brothers as he noted the
incongruity in the appearance of the company. "If anybody should
see us, they'd sure be likely to notice us with two of us all rigged out
to kill and the others not."
"I reckon it would be a good idea," returned his chief. "The people at
the farmhouse didn't seem to notice that our faces were smooth, at
least they didn't raise any yell about it, and they probably would
have if they'd caught on to the fact, so we'll have a better chance of
getting by in our usual costume."

Little time did it take the bandits to change but they carefully folded
the suits they took off and put them into their saddle bags for future
emergencies.
Their horses refreshed by the rest and the grass they had cropped,
Jesse gave the word to re-saddle and mount and they resumed their
cross-state ride.
Throughout the day they advanced, picking their course through the
woods, till along toward dusk their hunger decided them to halt near
a farmhouse where they determined to secure some food.
Leaving Cole with John to guard the horses, that the more
experienced man might steady the lad and parry any pertinent
questions should any inquisitive countryman stumble upon them, the
bandit-chieftain and Jim started for the house.
Keeping under cover of the bushes till they reached the road, they
scanned the buildings closely as they approached.
The unpainted boards, made grey by exposure to wind and rain,
bespoke the occupants as not overburdened with worldly goods. The
roof of a shed running from the house to the barn was sadly in the
need of repair, giving evidence of being on the verge of collapsing,
and as they turned into the yard a litter of pails, broken-down
wagons and all sorts of old rubbish suggested that the inmates were
shiftless as well as poor.
"Not very inviting," commented John, taking in the surroundings.
"You can't always judge by appearances," returned his chief.
And as the door was opened in response to his rap, his companion
conceded that he was right.
Looking at them with frightened eyes was a girl of possibly twenty
years, her beauty in startling contrast to the ragged calico wrapper
in which she was clad.

A mass of Titian hair, glorious despite its disorder, crowned a face
exquisite in its fine moulding and delicate colouring.
Her eyes, blue and innocent as a babe's, alone bore trace of the
poverty and want her environment proclaimed.
Blushing at the frank admiration in the faces of the men before her,
the girl seemed to realize the incongruity of her appearance and in a
tone of resentment demanded:
"What do you want?"
"We should like to get something to eat, if you will sell it to us,"
replied the bandit-chieftain.
"Food? You come here for food?" exclaimed the creature, and then
burst into a laugh, awful in its bitterness.
Surprised at her action, Jesse was about to speak when she went
on:
"We haven't enough for ourselves, let alone strangers. There's
nothing in the house but a little corn meal. Ma's in bed with a fever,
baby's ailing and they took our cow away from us today."
Then, as though ashamed for her rancor, she added: "But if we had
anything you'd be welcome."
His big heart ever warm for those in distress, as he heard of the
tribulations besetting the fair girl and her family, the world-famous
desperado became interested and when the loss of the cow was
disclosed, he uttered an ejaculation of anger that anyone should be
so cruel.
Something of what was passing through his mind showed in his eyes
and instead of closing the door, as she started to do, the girl looked
at him eagerly.
"Where are the men folks?" he asked.

Wincing as though she had been struck with a lash, the beauty drew
back.
"I beg pardon," hastily continued Jesse. "I didn't mean to add to
your anguish. I fancy I understand about them."
And the relief that his words brought to the blue eyes was ample
reward to the bandit-chieftain and he continued: "How would it be if
we should buy some food at the village, would you cook it for us?
We'll pay you for your trouble."
Scarce believing her ears, the girl looked at the outlaws as though
they were genii sprung from the earth, then exclaimed:
"I'll ask Ma. Won't you come in? You'll have to excuse the looks of
the house but I haven't felt much like tidying up."
Accepting the invitation eagerly, in the hope that he might get a
glimpse of the fever-stricken woman and the infant, Jesse entered.
But the sight that greeted his eyes made the yard seem clean and
well-kept, in comparison.
On one side of the room, which served as kitchen and bed-chamber,
stretched on a pallet of straw, lay a grey-haired woman, her thin
face and flushed cheeks evidencing all too clearly the ravages of the
malady with which she was afflicted.
And resting in the crook of an emaciated arm lay a baby, fussing and
whimpering, now and then crying:
"Mik. I wan' my mik."
In his interest in the twain on the sorry bed, the world-famous
desperado was oblivious to the rags, dishes, broken chairs and
battered stove that formed the rest of the furnishings.
"These men want to know if I'll cook 'em something to eat if they'll
buy it," announced the girl, dropping to her knees that she might
speak in the woman's ear.

"Law, child, I don' believe the stove'll draw," replied her mother,
when she understood the reason for the strangers' presence.
"Then we'll get food that won't need cooking," returned Jesse.
"You'll have to speak louder, Ma's deaf," declared her daughter.
"Well, you kin do as you please," rejoined the sick creature. "It may
draw and it may not."
"We'll take the chance," asserted the bandit-chieftain. "Jim, you and
Miss—"
"Shaw, Daisy Shaw," supplied the girl.
"You and Miss Shaw make out a list of what she needs at the store
while I see if I can't make her mother a bit easier." And kneeling
beside the bed of straw, he took out his medicine case with its
wonderful salves and lotions.
In reply to his questions, the bandit-chieftain learned that the
woman had been tossing with the fever for more than a week,
though not till the desertion of her husband and son, two days
before, had it become virulent.
The mention of the faithless scoundrel who had left her in want and
misery threw her into wild ravings.
"Does she have these spells often?" asked the great outlaw as he
hastily produced an opiate from his case.
"Most of the time. Oh, she takes on awful!" returned the girl whose
status as wife of the runaway son or sister, the bandits had not yet
ascertained.
But it was one thing to prepare the narcotic and another to
administer it.
At first the woman would not listen to the suggestion, protesting
that Jesse was but some miserable tool of her husband, sent by him

to poison her. And it required the combined efforts of the three to
reassure her. So weak was she from lack of nourishment and the
ravages of the fever that when she did swallow it the effect was
almost instantaneous, however.
By the time her mother was wrapped in the first sleep since her
abandonment by her husband, Daisy and Jim had completed the list.
"Have you thought of everything?" smiled Jesse as he noted the look
of fearsome eagerness on her face as she handed him the slip.
"That will do for the present," she replied, relieved that the number
of the wants had not appalled their benefactor.
"But there's not enough to last two days," protested the famous
desperado, glancing through the items. "Jim, go to the store—Miss
Shaw will direct you how to reach it, and order three times the
amount she's put down. Get a barrel of flour and a barrel of sugar,
too. Have someone drive the stuff back with you. Now hurry, I'm
hungry."
As his chum picked up his hat and departed, after receiving the
necessary instructions to reach the store. Daisy tried to thank the
generous stranger, but with a laugh, Jesse begged her not to
mention it and distracted her attention from his largess by
suggesting that she bathe her mother in a lotion he took from his
medicine case.
"She isn't my real mother," confided the girl, "only my mother-in-law.
I married Tom in New Orleans. He was a horse jockey at the time.
But he got to drinking, lost his job and we drifted up here—and now
he's left me."
"Good riddance, I should say," snapped Jesse. And by dint of clever
questioning, he drew from the girl the whole sad story of deception
and disgrace to which she and the sick woman had been brought by
the worthless father and son whose disappearance was due to some
transgression of the law.

His sympathy aroused, the famous desperado asked about the cow,
learning that she had been taken for a mortgage which was not due
for three days. Having a chance to sell her and believing that the
deserted woman could never raise the amount loaned, the
hardhearted farmer had driven the animal away.
Boiling with indignation at the injustice, Jesse demanded the man's
name.
"He's Hiram Rozier and he's awful rich," answered Daisy.
"I wonder if he is any relation to the Roziers in Ste. Genevieve?"
hazarded the bandit-chieftain.
"He has a brother who's president of or owns a bank."
"Ha! He is one of that tribe, eh?" hissed Jesse.
"I'll pay him a visit before I leave this town. You'll either have your
cow back or another in its place."
Something in the tone in which her "Prince Charming" uttered the
promise made Daisy look at him and the expression she saw on his
face caused her to shrink from him in terror.
But his paroxysm of rage lasted only a moment and when Jim
returned with the groceries and provisions half an hour later, they
were laughing and joking as they struggled to make the battered
stove do its duty.
When the supplies had been transferred from the wagon to the
house, Jesse bade his chum wait upon the girl while he transacted a
little business and without giving either Daisy or Jim a chance to
object, went from the house, jumping up beside the driver of the
grocery wagon whom he ordered to drive as fast as he could to the
home of Hiram Rozier.
The curiosity of the villager excited by the unheard-of order for
provisions for the Shaws, the fellow sought to learn Jesse's relation

to the family who were sneeringly alluded to as "poor white trash"
by their more prosperous neighbours.
Deeming it advisable to offer some explanation, the world-famous
desperado amused himself during the drive by unfolding a wondrous
tale of a long-lost sister, stolen by gypsies when she was a tot, found
in the person of Daisy Shaw.
"Here we be to Hiram's," announced the fellow, as he pulled up in
front of a big white house, the blinds on the front of which were all
shut tight. "Shall you want me any more?"
In the tone was a mixture of desire to impart the choice bit of gossip
of the returned brother to his cronies at the store and reluctance to
leave the stranger lest he miss something.
Smiling at it, Jesse replied:
"If you can, I should be obliged if you would listen to my
conversation with Mr. Rozier. I may want a witness."
Here was mystery indeed—the long-lost brother of Daisy Shaw
desiring a witness to a conversation with the richest man in town!
And in his haste to descend from the wagon, the clerk caught his
foot in the reins and would have fallen had not the bandit-chieftain
caught him.
Going boldly up to the front door, his companion following at a safe
distance, Jesse lifted the old fashioned brass knocker and let it fall
with a resounding rap.
In a few moments shuffling steps sounded and a man, whose face,
though older and topped with white locks, was a counterpart of the
banker's at Monegaw Springs, opened the door, demanding gruffly:
"What do you want?"
"Is this Mr. Hiram Rozier?" asked the famous desperado.
"It is."

Assured on this point, Jesse went to the point without any
preliminaries:
"You stole Mrs. Thomas Shaw's cow from her today, didn't you?"
"Stole her cow? No, sir! How dare you say such a thing?" thundered
the infuriated man.
"Don't raise your voice—unless you want your neighbors to hear,"
retorted his interrogator, coolly. "You did steal it and you know you
did! There was a mortgage on it but it wasn't due till day after
tomorrow.
"I've come to take back that cow!"
Dropping his bombastic manner, Hiram Rosier whined:
"I got it in a business way. I had a chance to sell her. Mrs. Shaw
couldn't pay the money even if it isn't due for three days. There's
nothing wrong about the transaction. I can't afford to lose fifteen
dollars and sixty cents when I—"
"Rubbish!" snapped Jesse. "Mrs. Shaw can pay the money. See,
here's a twenty-dollar bill. But she won't—with my permission.
"How much did you get for the cow?"
Too amazed by such treatment to think of refusing to reply, the old
man stammered:
"Forty dollars."
"Giving you a profit of practically twenty-five dollars, eh? Is that the
way you made all your money, stealing food from the mouths of
helpless women and children?"
"I won't listen to such abuse!" roared Hiram Rozier and he started to
shut the door in the great outlaw's face.

The latter had been expecting such a move, however, and quickly
reaching out his powerful right arm, seized the old man and yanked
him on to the porch, hissing:
"Oh, yes you will—and more too. I want you to get on this wagon
and drive with me to the man to whom you sold Mrs. Shaw's cow."
"And if I refuse?"
"I'll have you arrested for selling stolen property."
The humiliation and disgrace such a proceeding would bring upon
the name of Rozier decided the old man and he rejoined:
"Wait till I get my hat."
"Oh, no you don't. Do I look like a fool enough to let you go back
into the house? You put my hat on your head and come along."
Before the other could remonstrate, the world-famous desperado
had jammed his sombrero upon the white locks and was dragging
the old man toward the wagon.
Pinching himself to see if he were awake, the clerk followed and,
when all were on the seat, drove to the house named by Mr. Rozier.
Arrived, Jesse accompanied the unwilling moneylender to the door.
When the purpose of the visit was made known, the purchaser of
the cow at first refused to surrender her in return for the money he
had paid, finally declaring, however, that he would part with her for
fifty dollars.
Threatening to expose him should he decline to give the extra ten
dollars, Jesse forced the brother of the banker to pay it and with the
cow tied to the wagon, they drove back to his house.
The loss of the money rankling in his breast, as the team stopped in
front of his gate, Mr. Rozier exclaimed:

"If you'll give me the amount of the mortgage, I'll have it cancelled."
"Not much," retorted the bandit-chieftain. "You'll send the paper,
marked 'satisfied in full of all demands' 'round to Mrs. Shaw's tonight
before nine o'clock or she'll swear out a warrant against you!"
And without giving the terrified old man the opportunity to reply,
Jesse ordered the dumbfounded grocery clerk to drive on.

Chapter XV.
JESSE GETS HIS REVENGE.
Dismissing the clerk with a generous tip when they had returned to
the Shaw house, Jesse jumped from the wagon, untied the cow and
intended to wait till the fellow had driven away before calling to
Daisy to come out.
But the rattle of the wheels had been heard by the girl and Jim, and
they rushed to the door to learn who had arrived.
"Is that you, chief?" called the member of the Younger family,
unable to see in the darkness after leaving the lighted room.
Ere the great outlaw could reply, however, the cow gave a low
"moo," evidently glad to be home, and with a gasp of incredulous
delight, Daisy darted to the animal, threw her arms around its neck
and murmured:
"Is it really you, Topsey? How can we ever thank you, sir. Little Tom
will have his milk now—unless—unless—" and she stopped abruptly.
"Unless what, Miss Shaw?" inquired the bandit-chieftain.
"Unless Mr. Rozier only let her come back for the three days."
"Don't worry about that. Topsey, if that's her name, is yours for good
and all. Hiram Rozier will never trouble you about her."

Yet though the girl pressed him, he would say nothing about the
manner in which the animal had been recovered.
Leading the cow to her shed, Daisy bade Jim fetch a milk pail,
announcing that supper would be ready as soon as she had finished
the task.
But Jesse realized that Cole and John would be worried at the length
of time he and his companion were absent and, going into the
house, he helped himself to such articles of food as he thought his
pals would relish, then set out to rejoin them.
Their anxiety relieved by the return of their chief, the Youngers
listened interestedly to his account of the occurrences during his
absence, both of them stripping a bill from their rolls and asking him
to give them to the stricken family.
"Saddle the horses at nine o'clock," Jesse remarked after thanking
them for their contributions. "Jim and I'll be back as soon as we see
whether that brute Hiram sends the cancelled mortgage or not.
"Sit tight and don't take any chances."
Daisy and her companion had not been in the house long when the
world-famous desperado returned.
To his delight, the girl asked no embarrassing questions about his
absence, apparently considering it but natural that he should look
around, and no mention did she make of the missing edibles.
With a joy that was reward sufficient in itself, the baby took his milk,
being indulged with a double allowance in honour of the visit of
"Santa Claus" as Daisy called her benefactor.
On the pallet, Mrs. Shaw was sleeping peacefully.
Sitting down to the rickety table, the outlaws ate ravenously, for they
had been more than twenty-four hours without food.

Their hunger, appeased, Jesse suggested that on the morrow the girl
look for some cosy, clean rooms with a farmer who would keep
Topsey, leaving the ramshackle hovel for good and all.
The plan met with Daisy's approval till she remembered that such
comfort would cost money, but her disappointment was quickly
alleviated by the great outlaw.
"With proper care, I think Mrs. Shaw will throw off the fever," said
he, encouragingly. "You must have a doctor and give her and
yourself and the baby, too, plenty of nourishing food. Then, as soon
as your mother-in-law is able to travel, I think it would be a good
plan for you all to go down to your people, if you care to, in New
Orleans. If you don't I'll try to find a place for you somewhere. I'll
make it a point to come back and look you up in a couple of weeks,
anyhow. And in the meantime, if you'll accept a little present from
me, it'll keep you going till I see you again."
And as he concluded his speech, Jesse placed several crisp bank
notes on the table in front of the girl.
Fascinated by the sight of so much money, Daisy gazed at it for
several minutes, then with a little sigh, took one bill and pushed the
rest toward her "Prince Charming," murmuring:
"You're so good. I'll take this twenty dollars because it'll help pay the
doctor and feed little Tom. But I can't accept the rest. It wouldn't be
right. Why, look at all you've done for us, buying provisions and
getting back Topsey, and I don't even know your name."
"Never mind about that now," returned the great desperado,
awkwardly. "I'm always glad to assist people in want. You just put
that money in a safe place and don't think any more about it. Some
day, perhaps, you'll be able to help me and if you are, I shall count
upon your doing so."
Puzzled by the suggestion that so insignificant a person as she might
be of service to the strong, handsome man before her, the girl was

on the point of renewing her attempt to learn his identity when there
was a knock on the door.
Exchanging hurried glances, the bandit-chieftain and Jim rose to
their feet, crossing the room toward a window, their hands ready to
whip out their six-shooters should the summons be from some of
their enemies.
Unheeding the significant precaution, Daisy went to the door and
opened it.
"Here's a letter from Mr. Hiram Rozier," piped a boy's voice. "He says
he hopes Mrs. Shaw's better."
But the girl vouchsafed no reply, simply taking the envelope and
shutting the door in the messenger's face.
"What can this be?" she exclaimed, turning the missive over and
over in her hands as though trying to learn its contents through the
cover.
"Open it and find out," smiled Jesse. "I don't believe it's loaded."
Mechanically Daisy obeyed, drawing forth a carefully folded piece of
paper.
"Why, it's the mortgage on Topsey and it says 'satisfied in full of all
demands' on it. This is more of your work, sir," she cried, raising her
happy face to the great outlaw's.
"So I fancy. I thought the old duffer would listen to reason. And now
that everything's all right, we must go."
Finding that her entreaties were of no avail, the girl insisted upon
their taking lunch with them and after Jesse had showed her about
some medicines he left for the sick woman, with many expressions
of her gratitude for their surprising generosity, Daisy watched them
till they were lost in the darkness of the night.

For several rods the two outlaws walked on in silence, then the chief
exclaimed:
"I sure am glad we happened to strike that house."
Attributing the statement to his sympathetic nature, Jim praised his
bounty.
But when they rejoined Cole and John, he was made aware that it
was prompted by other causes as well.
"The old curmudgeon sent back the mortgage, cancelled,"
announced Jesse excitedly. "I've thrown a jolt into old Hiram that he
won't forget till his dying day. Now for the other Rozier! Come on.
We must be in Ste. Genevieve day after tomorrow."
Realizing that the task would mean hard riding, the bandits lost no
time in getting under way, passing through the town that Jesse
might point out where the lion he had "bearded in his den" lived, to
his fellows.
Twice during the night, the desperadoes were startled by the sound
of rapid hoof-beats and reined into the bushes at the side of the
road to discover who was riding so late.
The first one proved to be a solitary horseman coming from the
direction in which they were going, but the second caused them
deep concern.
Five men, with rifles carried across their saddle pommels, ready for
instant use, dashed along the highway, bound eastward as were the
bandits.
Never uttering a word, the men leaned forward the better to
penetrate the darkness ahead.
"Do you suppose they're after us or are they just out on a little game
of their own?" whispered Jim as the hoof-beats died away in the
distance.

"Goodness only knows," returned the world-famous desperado.
"They mean business, whoever they are. Just keep your ears open."
Fearing an ambush, the outlaws rode with the utmost caution,
carefully examining any dark places along the highway before they
entered.
The possibility that they were officers sent out by Hiram Rozier was
discussed but no decision could they come to and it was with
feelings of relief that they watched the day dawn.
Pausing in a dense copse for breakfast, they devoured the lunch
provided by Daisy and took turns at sleeping till about ten o'clock
when they once more took up their course.
Riding in the shelter of the woods, the outlaws covered many miles
before sundown when they again rested.
Forced to be content with smoking their pipes and tightening their
belts in lieu of supper, they stopped only long enough to feed their
mounts, then entered upon the final stage of their cross-state dash.
In contrast to the two previous nights, the moon rose clear, enabling
them the better to see the roadway and thus make faster time.
No untoward incidents occurred. Not a traveller did they meet and at
six o'clock in the morning they rode into Ste. Genevieve.
Founded by the Catholics in the early part of the eighteenth century,
the town had grown in wealth and population steadily. The old
French families had imparted a courtliness and refinement that
distinguished it from other villages when the horde of home-seekers
flocked across the "Big Muddy," lured by dreams of wealth and
happiness to be found beyond its shores.
With a thrift inherited from their forebears, the inhabitants saved
their money and Jim had not been wrong when he had declared that
Rozier's bank usually carried upwards of $100,000 in its vaults.

The air was redolent with the fragrance of Spring as Jesse, Jim, John
and Cole rode through the streets, their minds bent upon a crime
against the citizens.
Stopping at the first livery stable to which they came, the outlaws
ordered their horses fed and inquired the way to the nearest good
restaurant.
It was the belief of the world-famous desperado that men were
more daring with their stomachs full than when hunger gnawed at
them, and never did he undertake any of his sensational raids
without eating first, when possible.
Being directed to a first-class eating place, they laughed and chatted
during their breakfast and no one who saw them would have
thought they contemplated perpetrating any foul deed.
Purchasing cigars when they had finished their meal, the quartette
sauntered out onto the street to find where the Savings Association
Bank was located and to look over the building, its approaches and
entrances.
In the northern end of the town they discovered it, its name
announced by a gold-lettered sign with black background at the top
of a one-story building and in smaller letters on the windows.
"What luck," chuckled Jesse as they walked past the bank. "It's right
on a corner. You and Jim, John, can wait in this side street. There
are nothing but dwelling houses on it, that I can see. By looking
through the windows, you can see anybody passing on the main
thoroughfare and no inquisitive attention will be attracted to you as
there would if you sat your horses in front of the bank.
"When we go back, we'll notice what time it opens. The earlier we
turn the trick, the less risk we'll run of being interrupted."
Not just beyond the building did the bandits stop and turn, however.
Too thoroughly versed in the art of avoiding attention when they

wished to, they walked half a mile beyond the bank, crossed the
street and returned on the opposite side.
"Get a look at the cashier's cage," whispered their chief as they drew
near.
In the most casual manner, the desperadoes glanced toward the
institution, then turned their eyes upon something else.
But in the brief space they had learned what they wished.
White enamelled letters announced that the banking hours were
from nine till twelve in the morning and from two till four in the
afternoon, and on Saturday evenings from seven till eight.
The desk of the cashier was at the end of the counter next to the
side street, evidently so placed that the official might converse with
customers without being heard by any others who might be in the
bank.
And directly behind the desk the massive steel doors of the vault
were visible.
"It's only seven-thirty," declared Jesse, looking at his watch when
they had passed the bank. "Let's drop into the hotel and look at
yesterday's newspapers. I'd lake to see what they have to say about
the shindy at the Springs."
No better way of spending the hour and a half they were forced to
wait suggesting itself, the quartette entered the village Inn and were
soon engrossed in the fantastic report of Jesse's escape from the
health resort.
Beyond the usual, exaggerated account of the number of his
companions, there was nothing that caused them concern.
But the perusal served as an excellent "kill time" and it was with a
start that the great outlaw noticed the hands on the Inn clock
pointing to quarter before nine.

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Peptidomics Methods and Protocols 1st Edition Mikhail Soloviev (Auth.)

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