The mass spectrometry facility currently has two instruments, a MALDI-TOF mass spectrometer (MALDI microMX, Waters) and a nanospray linear ion trap mass spectrometer (LTQ XL, Thermo). In FY2013, the mass spectrometry facility within CPTR collaborated in 34 different projects, with more than 1200 samples processed and analyzed. Those projects and samples have spanned several different uses of mass spectrometry, such as identification of protein interactors, identification of post-translational modifications, and quantitative comparison of proteomes. Many of the projects are ongoing, but thus far, all have produced very interesting results for the collaborator and suggested specific hypotheses to be tested. In the past year, two studies incorporating results obtained in the mass spectrometry facility have been published. In addition, several other projects are nearing completion and will be prepared as manuscripts for publication. The first study, in Nature Medicine, is a collaboration with multiple investigators within and without NIH. In this study, the level of Factor-VIII (F8) mRNA and intracellular protein in B-lymphoblastoid cells and liver biopsies from healthy and subjects with an intron-22-inversion (I22I) in the F8 gene. Among individuals with hemophilia-A, those that have missense mutations in the F8 gene lack antigenically-cross reactive material (CRM) in their plasma, complicating treatment via Factor-VIII supplementation. Although I22I in the F8 gene, constituting approximately 50% of patients with severe hemophilia-A, should similarly lead to a lack of CRM in the plasma, only 20% of these patients actually develop neutralizing antibodies against replacement Factor-VIII. In I22I subjects, the presence of two F8-exon-containing mRNAs were observed, which together include all 26 F8 exons. Furthermore, these mRNAs lead to expression of the entire primary amino acid sequence of Factor-VIII as two non-secreted polypeptide chains. Mass spectrometry was used to confirm the presence of these two Factor-VIII polypeptides. In addition, a pharmacogenetic algorithm was developed that permits the stratification of neutralizing antibody risk for sub-populations by predicting immunogenicity using, as input, the number of putative T-cell epitopes in the infused Factor-VIII and the competence of MHC-Class-II molecules to present such epitopes. In the second study, a collaboration with the laboratory of Dr. Michael Maurizi in the Laboratory of Cell Biology, mass spectrometry analysis was used to investigate the N-degradome of E. coli. In Gram-negative bacteria and eukaryotes, the N-end rule is a conserved mechanism for targeting proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In E. coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. To better understand the mechanisms that produce N-degrons in proteins and the frequency of their occurrence, mass spectrometry was used to identify proteins that interact with ClpS. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates. This work has been published in the Journal of Biological Chemistry. Overall, the expertise of the facility is being widely used to apply mass spectrometry to multiple research projects. As stated above, the facility has been involved in 34 different projects;these span 21 different CCR investigators, including 3 tenure-track investigators. Among these projects are several similar to the ones published, in which the identity of particular proteins or protein interactors are investigated. In addition to these, there are multiple projects to identify sites of post-translational modification, including phosphorylation, ubiquitination, acetylation, and modification with small molecule inhibitors. Finally, the resource is working on large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify the proteome of an organelle, fluid, or protein interaction network.
|Humbard, Matthew A; Surkov, Serhiy; De Donatis, Gian Marco et al. (2013) The N-degradome of Escherichia coli: LIMITED PROTEOLYSIS IN VIVO GENERATES A LARGE POOL OF PROTEINS BEARING N-DEGRONS. J Biol Chem 288:28913-24|