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Recent studies utilizing transcriptomics, metabolomics, and bottom up proteomics have identified molecular signatures of kidney allograft pathology. Although these results make significant progress toward non-invasive differential diagnostics of dysfunction of a transplanted kidney, they provide little information on the intact, often modified, protein molecules present during progression of this pathology. Because intact proteins underpin diverse biological processes, measuring the relative abundance of their modified forms promises to advance mechanistic understanding, and might provide a new class of biomarker candidates. Here, we used top down proteomics to inventory the modified forms of whole proteins in peripheral blood mononuclear cells (PBMCs) taken at the time of kidney biopsy for 40 kidney allograft recipients either with healthy transplants or those suffering acute rejection. Supported by gas-phase fragmentation of whole protein ions during tandem mass spectrometry, we identified 344 proteins mapping to 2,905 distinct molecular forms (proteoforms). Using an initial implementation of a label-free approach to quantitative top down proteomics, we obtained evidence suggesting relative abundance changes in 111 proteoforms between the two patient groups. Collectively, our work is the first to catalog intact protein molecules in PBMCs and suggests differentially abundant proteoforms for further analysis.

Pilot Project #1--the identification and characterization of human histone H4 proteoforms by top-down MS--is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10(-13) to 10(-105). Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.