Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global Post-Translational Modification Discovery Database

Abstract

Complex human biomolecular processes are made possible by the diversity of human proteoforms. Constructing proteoform families, groups of proteoforms derived from the same gene, is one way to represent this diversity. Comprehensive, high-confidence identification of human proteoforms remains a central challenge in mass spectrometry-based proteomics. We have previously reported a strategy for proteoform identification using intact-mass measurements, and we have since improved that strategy by mass calibration based on search results, the use of a global post-translational modification discovery database, and the integration of top-down proteomics results with intact-mass analysis. In the present study, we combine these strategies for enhanced proteoform identification in total cell lysate from the Jurkat human T lymphocyte cell line. We collected, processed, and integrated three types of proteomics data (NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up) to maximize the number of confident proteoform identifications. The integrated analysis revealed 5950 unique experimentally observed proteoforms, which were assembled into 848 proteoform families. Twenty percent of the observed proteoforms were confidently identified at a 3.9% false discovery rate, representing 1207 unique proteoforms derived from 484 genes.

Qualitative and quantitative top-down proteomics of human colorectal cancer cell lines identified 23000 proteoforms and revealed drastic proteoform-level differences between metastatic and non-metastatic cancer cells

“…One important value of TDP is its capability for delineation of various proteoforms from the same gene (proteoform family). [42] Figure 3E shows one example of Calmodulin-1 (CALM1) proteoform family. Calmodulin-1 modulates many enzymes (kinases and phosphatases), ion channels, and many other proteins by calcium-binding.…”

Spritz: A Proteogenomic Database Engine

“…The second dataset used to assess the identification of modified variant sites is a phosphoproteomic analysis of U2OS cells 21 , along with U2OS RNA-Seq data obtained from the Human Protein Atlas 22 (SRR629563 in GEO SRA). Finally, a recently published top-down proteomics dataset for Jurkat was used to demonstrate the identification of variant-containing proteoforms 5 .…”

“…Top-down mass spectrometry (MS) is a proteomic method that can directly analyze proteoforms 2,3 , and bottom-up MS analysis can approximate this type of study by analyzing peptides from these proteoforms. We previously demonstrated how using a database annotated with modifications discovered in bottom-up proteomic analysis expands the number of proteoform identifications 4,5 . In this work, we demonstrate how creating databases with full length proteoform sequences containing variations can improve the identification of amino acid sequence variations, allow identification of modified variant peptides, and enable the identification of variant proteoforms.…”

Proteoform-selective imaging of tissues using mass spectrometry

“…[6][7][8][9][10][11][12][13] Several databases have been developed as a community resource for proteoform identification. [14][15] [16] Several studies used top-down proteomics for the discovery of disease biomarkers. [17] For example, differences in the expression levels of several KRAS proteoforms have been associated with cancer cell proliferation.…”