Search

Accurate assignment of monoisotopic precursor masses to tandem mass spectrometric (MS/MS) data is a fundamental and critically important step for successful peptide identifications in mass spectrometry based proteomics. Here we describe an integrated approach that combines three previously reported methods of treating MS/MS data for precursor mass refinement. This combined method, 'integrated post-experiment monoisotopic mass refinement' (iPE-MMR), integrates steps (1) generation of refined MS/MS data by DeconMSn; (2) additional refinement of the resultant MS/MS data by a modified version of PE-MMR; and (3) elimination of systematic errors of precursor masses using DtaRefinery. iPE-MMR is the first method that utilizes all MS information from multiple MS scans of a precursor ion including multiple charge states, in an MS scan, to determine precursor mass. With the combination of these methods, iPE-MMR increases sensitivity in peptide identification and provides increased accuracy when applied to complex high-throughput proteomics data. 10.1021/ac101388b

Chemical cross-linking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of cross-linked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact cross-linkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under conditions where the native complex structure is stable. In this study, we present a novel compact cross-linker that contains two distinct features: (1) an alkyne tag and (2) a small molecule detection tag (N[O.sub.2]) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the cross-linked peptides after proteolytic cleavage and coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small N[O.sub.2] moiety provides a secondary means of detecting cross-linked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this cross-linker, which we termed CLIP (click-enabled linker for interacting proteins) using ubiquitin. The enrichment capability of CUP is demonstrated for cross-linked ubiquitin in highly complex E. coli cell lysates. Sequential collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and electron transfer dissociation (ETD)-MS/MS of intercross-linked peptides (two peptides connected with a cross-linker) are also demonstrated for improved automated identification of cross-linked peptides.

The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the 'state' of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography mass spectrometry (LC/MS) analysis of intact phosphoproteins. The technology platform builds upon an integration of bottom-up and top-down approaches that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection. As the use of conventional RPLC systems for phosphopeptide identification has proven challenging due to the formation of metal ion complexes at various metal surfaces during LC/ MS and ESI-MS analysis, we have developed a 'metal-free' RPLC-ESI-MS platform for phosphoprotein characterization. This platform demonstrated a significant sensitivity enhancement for phosphorylated casein proteins enriched from a standard protein mixture and revealed the presence of over 20 casein isoforms arising from genetic variants with varying numbers of phosphorylation sites. The integrated workflow was also applied to an enriched yeast phosphoproteome to evaluate the feasibility of this strategy for characterizing complex biological systems and revealed ~16% of the detected yeast proteins to have multiple phosphorylation isoforms. The intact protein LC/MS platform for characterization of combinatorial post-translational modifications (PTMs), with special emphasis on multisite phosphorylation, holds great promise to significantly extend our understanding of the roles of multiple PTMs on signaling components that control the cellular responses to various stimuli.

Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1100 peptides and their ~200 protein-substrate origins we obtain evidence for new insights into the proteome-wide proteinselective degradation in yeast cells. This evidence shows that the yeast cytoplasm is the largest pool for the degradation of proteins with both biochemical and geometric specificities, whereas the yeast nucleus seems to be a proteolysis-inert organelle under the condition studied. Yeast V-ATPase subunits appear to be degraded during their disassembly, and yeast mitochondrial proteins functioning as precursors, transport carriers, and gates are preferentially degraded. Ubiquitylation may be unnecessary for the proteasomal degradation of yeast cytoplasmic regulatory and enzyme proteins according to our observations. This study shows that the intracellular peptides are informational targets for directly probing the protein degradation-involved molecular mechanisms and cell biology processes.