High‐sensitivity analysis of specific peptides in complex samples by selected MS/MS ion monitoring and linear ion trap mass spectrometry: Application to biological studies

Abstract

Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification.

Studies on the Reaction of Nitric Oxide with the Hypoxia-Inducible Factor Prolyl Hydroxylase Domain 2 (EGLN1)

“…Although it has been questioned whether it would be possible to detect labile S-nitrosylation 30 by MS, recently, "direct" measurement of protein S-nitrosylation by LC-MS analyses has been reported. [31][32][33] In the present study, apparent S-nitrosylation of four cysteines, Cys201, Cys208, Cys302, and Cys323/ Cys326, was detected ( Fig. 3 and Fig.…”

The Intracellular Interactome of Tetraspanin-enriched Microdomains Reveals Their Function as Sorting Machineries toward Exosomes

“…For peptide identification, the LTQ was operated in a data-dependent MS/MS mode using the 15 most intense precursors detected in a survey scan from 400 to 1,600 m/z, whereas for quantification of 16 O/ 18 O labeled peptides, the LTQ was operated in data-dependent ZoomScan and MS/MS switching mode (23,24). Protein identification was carried out as described previously (23) using the SEQUEST algorithm (Bioworks 3.2 package, Thermo Finnigan).…”

The Leukocyte Activation Receptor CD69 Controls T Cell Differentiation through Its Interaction with Galectin-1

“…After staining with Coomassie blue, gel samples were digested in gel with trypsin, and the resulting peptides were identified by mass spectrometry (MS) as described previously (23). Selected tandem MS (MS/MS) ion monitoring was performed as described previously (24). Primary antibodies for immunoblotting were goat polyclonal anti-Gal-1, anti-Gal-3, and anti-Gal-9 (R&D Systems, MN) and monoclonal anti-Gal-1 (Vector Laboratories, CA).…”