Julian Vasilescu

Large‐scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large‐scale study of protein–protein interactions in human cells using a mass spectrometry‐based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large‐scale immunoprecipitation of Flag‐tagged versions of these proteins followed by LC‐ESI‐MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross‐validated using previously published and predicted human protein interactions. In‐depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

Glycoproteomic Reactor for Human Plasma

We describe the development of a glycoproteomic reactor that combines multiple biochemical and chemical protein processing into a single device for the study of N-glycosylated proteins. The glycoproteins are first enriched by concanavalin A affinity chromatography and then transferred onto and efficiently processed in the glycoproteomic reactor. This glycoproteomic reactor combines protein concentration and purification, disulfide bond reduction, peptide-N-glycosidase-mediated (18)O-labeling and deglycosylation, alkylation, tryptic digestion and pH based fractionation in a device that has an interstitial volume (reaction volume) of approximately 1 microL. We demonstrated the potential of the glycoproteomic reactor using human plasma. Under stringent criteria, 82 unique glycopeptides representing 41 unique glycoproteins were identified from as little as 5 microL of human plasma. Our glycoproteomic reactor reduces the sample processing time to less than 1.5 h, reduces the reagent consumption while providing over 1000-fold concentration of the sample, provides efficient removal of high concentration of glycan buffer, and, finally, allows both glycopeptides and nonglycosylated tryptic peptides to be analyzed by the mass spectrometer which provides much greater protein coverage and more reliable identifications.

PCSK4-null sperm display enhanced protein tyrosine phosphorylation and ADAM2 proteolytic processing during in vitro capacitation

OBJECTIVE To study the molecular basis for the accelerated capacitation rate in PCSK4-null sperm. DESIGN Comparative and controlled experimental research study. SETTING Academic medical institute. ANIMAL(S) Male mice C57BL/6J wild-type or null congenics for the Pcsk4 allele. INTERVENTION(S) Cauda and epididymal sperm were capacitated for varying times. MAIN OUTCOME MEASURE(S) Differences in sperm protein tyrosine phosphorylation and proteolytic processing of sperm-egg ligands ADAM2 and ADAM3. RESULT(S) The PCSK4-null sperm proteins are hyper-tyrosine phosphorylated during capacitation. This hyperphosphorylation is dependent on protein kinase A (PKA), albumin, and calcium. There is also more ADAM2 proteolytic processing from a 46-kDa form of ADAM2 to a 27-kDa form in PCSK4-null sperm than in wild-type sperm. This processing is dependent on cholesterol efflux. CONCLUSION(S) Lack of PCSK4 is associated with quantitative changes in the phosphorylation and proteolysis of sperm proteins during capacitation; therefore, alterations in signal transduction and proteolytic processing during capacitation may underlie the fertilization incompetence of PCSK4-null sperm. More investigation is needed to determine how and to what extent these changes might contribute to the loss of fertilizing ability of PCSK4-null sperm.

Identification of lysines within membrane-anchored Mga2p120 that are targets of Rsp5p ubiquitination and mediate mobilization of tethered Mga2p90

Mga2p90 is an endoplasmic reticulum (ER)-localized transcription factor that is released from the ER membrane by a unique ubiquitin (Ub)-dependent mechanism. Mga2p90 mobilization requires polyubiquitination of its associating membrane-bound Mga2p120 anchor and subsequent Mga2p120-Mga2p90 complex disassembly that is mediated by ATPase Cdc48p and its heteromeric Ub-binding adaptor Npl4p-Ufd1p. Although previous studies have identified the Ub ligase (i.e., Rsp5p) and ligase-binding site on Mga2p120 that play a role in this process, the amino acids of Mga2p120 that are targets of ubiquitination and promote Mga2p90 mobilization are unknown. We have identified, using mass spectrometry analysis of in vitro ubiquitinated Mga2p120-Mga2p90 complex, that lysine residues 983 and 985 contained within the carboxy-terminal domain of Mga2p120 are Rsp5p-directed Ub-conjugation sites. Mutation of these residues as well as proximally located lysine 980 results in suppression of Mga2p120 ubiquitination in vitro and in vivo, inefficient liberation of Mga2p90 by Cdc48p(Npl4p/Ufd1p)in vitro, and ER retention of Mga2p in cells. Moreover, mga2Delta/spt23ts harboring Rsp5p binding and conjugation mga2 mutants express low OLE1 (an Mga2p90 target gene) transcripts and display reduced growth. We conclude that residues 980, 983, and 985 are targets of Rsp5p-induced polyubiquitination and mediate Cdc48p(Npl4p/Ufd1p)-dependent Mga2p90-Mga2p120 separation and Mga2p90 mobilization.