Ultramicroanalysis of Peptide Profiles in Biological Samples Using MALDI Mass Spectrometry

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Abstract

In the past few years, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has emerged as a powerful tool for the direct analysis of peptide profiles contained in single neuroendocrine cells, tissue biopsies, as well as in releasates (after a simple sample clean-up). These studies were performed in both invertebrate (the pond snail) and vertebrate (xenopus and rat) species. The present article provides an overview of these data with a special emphasis on the sample handling required for each application.

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Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

M Wilm, A. Shevchenko, T Houthaeve … (1996)

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

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Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins.

Myron S Cohen, B Chait (1996)

Sample-matrix preparation procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are observed when the matrix 4-hydroxy-alpha-cyanocinnamic acid is used for analyzing complex mixtures of peptides and proteins. The discrimination effects are found to be strongly dependent on the sample-matrix solution composition, pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI analysis of biological samples. The results also indicate that there is a reverse-phase chromatographic-like dimension in the sample-matrix preparation procedures that can be exploited to optimize the analysis. The present work describes the conditions under which the majority of components of a complex mixture of peptides and proteins can be successfully measured.

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Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling.

W Geraerts, B Reinhold, J. Peter H. Burbach … (1997)

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

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Author and article information

Journal

Journal ID (publisher-id): EXN

Journal ID (nlm-ta): Nephron Exp Nephrol

Journal ID (doi): 10.1159/issn.1660-2129

Title: Cardiorenal Medicine

Publisher: S. Karger AG

ISBN: 978-3-8055-6765-7

ISBN: 978-3-318-00352-9

ISSN (Electronic): 1660-2129

Publication date Subscription-year: 1998

Publication date (Print): October 1998

Publication date (Electronic): 11 September 1998

Volume: 6

Issue: 5

Pages: 421-428

Affiliations

^a Department of Molecular Neurobiology, Research Institute Neurosciences, Vrije Universiteit, Amsterdam, The Netherlands, and ^b Department of Pharmaceutical Chemistry, Mass Spectrometric Facility, University of California, San Francisco, Calif., USA

Article

Publisher ID: 20551 Other ID: Exp Nephrol 1998;6:421–428

DOI: 10.1159/000020551

SO-VID: 8ad397d8-232e-4c6e-ab73-ada165cbde10

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Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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Ultramicroanalysis of Peptide Profiles in Biological Samples Using MALDI Mass Spectrometry

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Abstract

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Karger: Nephrology

Most cited references 4

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling.

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