A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.

Protein-protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.

Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures.

The considerable progress in high-throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the past decade has been fueled to a large degree by continuous improvements in instrumentation. High-throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times 2 times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker's yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 full width at half-maximum (fwhm), it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments.

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer.

Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within +/-2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collision-induced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications.

Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem mass tags and electron transfer dissociation.

The modification of Ser/Thr residues in proteins by addition of single O-linked N-acetylglucosamine (O-GlcNAc) moieties play an important role in cell regulation. However, understanding the cellular mechanisms that regulate O-GlcNAc glycosylation has been challenging due to the difficulty in detection and quantification of this modification. Mass spectrometry-based multiplex quantitative approaches have been successfully employed to measure relative phosphorylation levels using collisionally induced dissociation (CID). However, labile modifications such as O-GlcNAc are lost prior to fragmentation of the peptide backbone in conventional CID, often preventing correct peptide identification, localization of the modified site, and as a result, relative quantification. Compared to CID, Electron Transfer Dissociation (ETD) preserves labile post-translational modifications (PTMs), and allows direct mapping of peptide/protein modifications. This is the first report to assess the utility of combining multiplexed isobaric tandem mass tag (TMT) labeling and ETD for relative quantification of labile PTMs. ETD analysis of both labeled and unlabeled peptides from bovine alpha-crystallins pinpointed at least one O-GlcNAc containing modification site in each of the protein subunits, in addition to a multitude of other PTMs, including glycation, phosphorylation, and acetylation. Moreover, ETD of TMT(6) labeled peptides produced four unique reporter ions that could be used for relative quantification. TMT reporter ion ratios measured by ETD had similar accuracy and precision as those obtained by conventional CID techniques. When applied to glycosylated or otherwise modified peptides, ETD was the only dissociation method which consistently provided confident sequence identification, PTM localization, and quantitative information, all in the same spectrum. This suggests that ETD-based workflows can be complementary to traditional CID approaches when used for simultaneous qualitative and quantitative analysis of modified peptides.