Abstract

Peptidomics, the analysis of the peptide content of cells or tissues, can be used to study proteases in several ways. First, nearly all of the peptides detected in cells and tissues are proteolytic fragments of proteins. Analysis of the peptides therefore provides information regarding the proteolytic activities that occurred to generate the observed peptides. The use of quantitative peptidomic approaches allows the comparison of relative peptide levels in two or more different samples, which enables studies examining the consequences of increasing proteolytic activity (by enzyme activation or overexpression) or reducing proteolytic activity (by inhibition, knock down, or knock out). Quantitative peptidomics can also be used to directly test the cleavage specificity of purified proteases. For this, peptides are purified from the tissue or cell line of interest, incubated in the presence of various amounts of protease or in the absence of protease, and then analyzed by the quantitative peptidomics approach. This reveals which peptides are preferred substrates, which are products, and which are not cleaved. Collectively, these studies complement conventional approaches to study proteolytic activity and allow for a more complete understanding of an enzyme's substrate specificity. This unit describes the use of quantitative peptidomics in the analysis of the biological peptidome as well as in the in vitro analysis of peptidase activity. Curr. Protoc. Protein Sci. 65:18.13.1?18.13.12. © 2011 by John Wiley & Sons, Inc.

Keywords: proteomics; protease; mass spectrometry

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Materials

Basic Protocol 1: Preparation and Analysis of Peptides by LC/MS

Materials

Mice
0.1 M hydrochloric acid (from 6 N, sequanal grade, constant boiling, Pierce)
0.4 M NaH₂ PO₄ (Sigma‐Aldrich)
TMAB‐NHS compounds (synthesis of these compounds has been described in Morano et al., )
1.0 M NaOH (Sigma‐Aldrich)
Dimethyl sulfoxide (Sigma‐Aldrich)
NH₂ OH⋅HCl (Sigma‐Aldrich)
Glycine (Sigma‐Aldrich)
Acetonitrile, HPLC grade (Fisher Scientific)
Trifluoroacetic acid (Pierce)
Formic acid

Low‐retention microcentrifuge tubes (Eppendorf)
Milli‐Q distilled water system (Millipore)
Ultrasonic processor (e.g., model W‐380, Ultrasonic Inc.)
Hydrion pH papers, 8.0‐9.5 (Micro Essential Laboratory)
Centrifugal filter devices (Amicon Ultra 4‐ml Ultracel, 10,000 MWCO, Millipore)
PepClean C‐18 spin column (Pierce)
LC/MS with C18 trapping column (e.g., Symmetry C18 trapping column, 5‐µm particles, 180‐µm i.d. × 20 mm, Waters) and separating column (e.g., BEH 130 C18 column, 1.7‐µm particles, 100‐µm i.d. × 100 mm, Waters)

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.

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Figures

Figure 18.13.1 Method of labeling peptides with TMAB tags. (A ) The structure of the TMAB tags is shown. The N ‐hydroxysuccinimide (NHS) moiety is replaced by the N‐terminal or lysine side chain amines upon reaction with peptides. Five different isotopic forms of the label can be made containing different numbers of hydrogen, deuterium, ¹² C, or ¹³ C in the methyl (Me) groups. (B ) To identify peptides and compare their levels among multiple tissues or treatments, peptides are extracted from tissues or cells and labeled directly with isotopic TMAB reagents. These peptides are then pooled and analyzed by LC/MS. (C ) To directly test the ability of proteases to cleave a variety of peptides, the peptides are first extracted from an appropriate biological sample (such as a tissue or cell population) and aliquots are digested with different amounts of a proteolytic enzyme (CONC 1‐4). The digests are differentially labeled with TMAB reagents, pooled, and analyzed by LC/MS.

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Figure 18.13.2 Representative peptide substrates and products identified by quantitative peptidomics upon incubation with a peptidase. Peptides were extracted from mouse brain, digested with different amounts of purified carboxypeptidase A6, labeled with isotopic TMAB tags as indicated, and analyzed by LC‐MS/MS. Examples of representative data are shown for (A ) good substrates, (B ) weak substrates, (C ) non‐substrates, and (D ) products. Many additional substrates and products were detected in this analysis (Lyons and Fricker, ).

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Literature Cited

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Peptidomic Approaches to Study Proteolytic Activity

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation and Analysis of Peptides by LC/MS

Figures

Literature Cited

Peptidomic Approaches to Study Proteolytic Activity

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation and Analysis of Peptides by LC/MS

Figures

Videos

Literature Cited