Screening for disulfide‐rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Abstract

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.

Cysteine tagging for MS‐based proteomics

“…For instance, the reaction with iodoacetamide served for cysteine counting of complex peptide extracts (Neitz et al, 2001). The mass spectrometric analysis is performed before and after the chemical derivatization.…”

Electrochemical multi-tagging of cysteinyl peptides during microspray mass spectrometry: numerical simulation of consecutive reactions in a microchannel

“…Counting the cysteine groups in a peptide using the isotopic signature of a specific tag for thiol 12,15 or the differential analysis of unlabelled and labelled cysteinyl samples 16 can be used to significantly improve protein identification by database searching. Recently, the electrochemically induced tagging of peptides by probes electrogenerated at the microband electrode of a microspray emitter 17 have enabled the on-line counting of cysteine units in peptides 18 to improve the processes of identification of model proteins.…”

Current chemical tagging strategies for proteome analysis by mass spectrometry

“…Alternatively, MS spectra can be acquired before and after alkylation and Cys residues can be identified from the mass shifts that are observed [95][96]. The presence of cysteine in a peptide can be used as a constraint in database searches, increasing confidence in the results and/or reducing search times.…”