Quantifying Homologous Proteins and Proteoforms

Abstract

Many proteoforms-arising from alternative splicing, post-translational modifications (PTM), or paralogous genes-have distinct biological functions, such as histone PTM proteoforms. However, their quantification by existing bottom-up mass-spectrometry (MS) methods is undermined by peptide-specific biases. To avoid these biases, we developed and implemented a first-principles model (HIquant) for quantifying proteoform stoichiometries. We characterized when MS data allow inferring proteoform stoichiometries by HIquant and derived an algorithm for optimal inference. We applied this algorithm to infer proteoform stoichiometries in two experimental systems that supported rigorous bench-marking: alkylated proteoforms spiked-in at known ratios and endogenous histone 3 PTM proteoforms quantified relative to internal heavy standards. When compared with the benchmarks, the proteoform stoichiometries interfered by HIquant without using external standards had relative error of 5-15% for simple proteoforms and 20 -30% for complex proteoforms. A HIquant server is implemented at: https://web.northeastern.edu/slavov/2014_HIquant/ https://www.mcponline.org Downloaded from FIG. 3. HIquant accurately infers stoichiometries and confidence intervals across PTM site occupancies of histone 3.A, Histone 3 peptides were quantified by SRM across 7 perturbations, and the fractional site occupancies for K4 methylation estimated by two methods: Estimates inferred by HIquant without using external standards are plotted against the corresponding estimates based on MasterMix external standards with known concentrations (29). Each marker shape corresponds to the PTM site(s) shown in the legend; methylation is denoted with "me" and acetylation with "ac" followed by the number of methyl/acetyl groups. B, The validation method from (a) was extended to another set of more complex fractional site occupancies on K9 methylation and K14 acetylation.

Analyzing Ribosome Remodeling in Health and Disease

“…[83,84] These biases can be very significant when estimating absolute protein abundances (due to differences in peptide flyability, a collective term describing efficiency of ionization and detection), and these biases may cancel out in relative protein quantification. [85,86]…”

“…This idea of canceling out biases can be extended by first-principle models, such as HIquant, to allow quantifying stoichiometry between different proteins independent from peptide specific biases. [86] Such an approach is likely to be particularly fruitful for quantifying RP preforms originating from different prologues, alternative splicing events or post-translational modifications. [14,86,88] In bottom-up proteomics, cells can be lysed and proteins extracted by a variety of methods.…”

“…[86] Such an approach is likely to be particularly fruitful for quantifying RP preforms originating from different prologues, alternative splicing events or post-translational modifications. [14,86,88] In bottom-up proteomics, cells can be lysed and proteins extracted by a variety of methods. [89][90][91] Then proteins are digested to peptides, which are separated via liquid chromatography (LC) and ionized through electrospray ionization or matrix-assisted desorption/ionization.…”

Splicing isoform-specific functional genomic in cancer cells

“…Of note, the QPsi formula doesn't include any normalizing gene. Although antisense oligonucleotides should have a minimal off-target effect, confirmation by unbiased and genome-wide methodologies such as mass spectrometry and RNA sequencing have not been reported [56,57].…”

DO-MS: Data-Driven Optimization of Mass Spectrometry Methods

“…Analytical methods combining liquid chromatography and tandem mass-spectrometry (LC-MS/MS) allow for unparalleled identification and relative quantitation of the protein components of biological systems. [1][2][3][4] Advances in LC-MS/MS have enabeled analysis of protein complexes and their functions, [5][6][7][8][9] regulation of protein synthesis and alternative RNA translation, 10,11 rare cells in blood, 12,13 and protein conformations. [14][15][16] The increasing sensitivity, [17][18][19][20][21][22][23] throughput, and robustness 24 of LC-MS/MS set the stage for quantifying thousands of proteins across many thousands of single cells, providing data with transformative potential for biomedical research.…”