Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Towards Allele-Specific Dynamic Proteomics: Measuring Protein Synthesis and Turnover Using Heavy Water Labelling and Peptide Mass Spectrometry.

Deery1,  Michael J., Riva2,  Alessandra De, Prevosto2,  Claudia, McDonald2,  Sarah, Busch2,  Robert

Centre for Proteomics, University of Cambridge
Dept of Medicine, University of Cambridge

Background:

Genome-wide association studies have uncovered structural and promoter polymorphisms associated with autoimmune disease and confirmed long-established associations with major histocompatibility complex (MHC) alleles. The underlying mechanisms often remain unclear; allelic differences in protein synthesis, folding, maturation, and turnover are of interest. Numerous techniques exist for measuring protein synthesis and turnover, but they suffer drawbacks when studying allelic effects in biologically relevant systems. Here, we have combined stable isotope labelling using heavy water (2H2O) with peptide mass spectrometry to devise a biosynthetic labelling approach suitable for allele-specific dynamic proteomics.

Methods:

MHC proteins were studied as a model system with disease-relevant isotypic and allelic variation. Antigen-presenting cells (APCs), such as B-lymphoblastoid cell lines, murine splenocytes, or short-term cultures derived from peripheral blood, were subjected to continuous labelling with 2H2O. MHC proteins were isolated by immunoprecipitation and SDS-PAGE, fragmented with trypsin, and peptides identified using LC/MS/MS. Selected peptides were analysed for 2H incorporation by LC/MS. Labelling patterns were interpreted using mass isotopomer distribution analysis and used to calculate fractional protein synthesis as a function of labelling time.

Results:

Analysis of tryptic peptides from murine or human MHC class I or class II proteins enabled protein identification and measurement of biosynthetic label incorporation, using small samples of a variety of APC types. Measured peptide mass isotopomer distributions were consistent with 2H label incorporation from 2H2O at a fixed number of effective labelling sites and allowed calculation of fractional synthesis rates. Protein turnover was quantifiable after accounting for cell growth or turnover and changes in protein levels. Estimates of fractional protein synthesis from analysis of different peptide mass isotopomers, and from different peptides derived from the same protein, were internally consistent and congruent with literature estimates based on other methods. Well-labelled peptides were identified that distinguished selected MHC class I and class II isotypes or alleles, enabling allele-specific measurements of protein synthesis and turnover. Analysis of multiple peptides indicated that multiple amino acids contributed to label incorporation from 2H2O; algorithms for predicting labelling patterns from sequence are being refined.

Conclusions:

The safety, simplicity, and versatility of 2H2O labelling combine with the sensitivity, precision, and specificity of LC/MS to enable allele-specific measurements of MHC protein synthesis and turnover. The approach is readily extended to other proteins and can be applied to primary cells and in vivo. These capabilities will aid studies of protein dynamics as a link between genetic polymorphisms and mechanisms of pathogenesis. The ability of the technique to accommodate complex samples suggests potential for proteome-wide studies of protein dynamics.

To cite this abstract, please use the following information:
Deery, Michael J., Riva, Alessandra De, Prevosto, Claudia, McDonald, Sarah, Busch, Robert; Towards Allele-Specific Dynamic Proteomics: Measuring Protein Synthesis and Turnover Using Heavy Water Labelling and Peptide Mass Spectrometry. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :843
DOI: 10.1002/art.28611

Abstract Supplement

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