Selected Ion Extraction of Peptides with Heavy Isotopes and Hydrogen Loss Reduces the Type II Error in Plasma Proteomics
Journal
ACS Omega
Journal Volume
10
Journal Issue
1
Start Page
281
End Page
293
ISSN
2470-1343
Date Issued
2025-01-02
Author(s)
Jaimie Dufresne
Zhuo Zhen Chen
Pallvi Sehajpal
Peter Bowden
John G. Marshall
Abstract
Naturally occurring peptides display a wide mass distribution after ionization due to the presence of heavy isotopes of C, H, N, O, and S and hydrogen loss. There is a crucial need for sensitive methods that collect as much information as possible about all plasma peptide forms. Statistical analysis of the delta mass distribution of peptide precursors from MS/MS spectra that were matched to 63,077 peptide sequences by X!TANDEM revealed Gaussian peaks representing heavy isotopes and hydrogen loss at integer delta mass values of −3, −2, −1, 0, +1, +2, +3, +4, and +5 Da. Human plasma samples were precipitated in acetonitrile, and the resulting proteins were collected over a quaternary amine resin, eluted with NaCl, digested with trypsin, and analyzed by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with an orbital ion trap (OIT). Fragment spectra (MS/MS) generated from the OIT data were fit to human fully tryptic peptides by X!TANDEM, which led to the identification of 3,888 protein gene symbols represented by three or more peptides (n ≥ 3). The peptide counts to plasma proteins from experimental MS/MS spectra were corrected against 29 blank LC-ESI-MS/MS spectra and 30 million random MS/MS control spectra to yield 2,784 true positive proteins (n ≥ 3; q ≤ 0.01). Peptides identified by fragmenting ions with Gaussian heavy isotopes and hydrogen loss that were matched to known plasma proteins, such as albumin (ALB), were shown to be true positives and agreed with the peptide sequences identified in the monoisotopic peak. Accepting the ions from the monoisotopic peak alone (±0.1 Da) yielded only 382 plasma proteins (n ≥ 3; type I error q ≤ 0.01; type II error ∼86%). In contrast, accepting all ions within ±0.1 Da around the hydrogen loss, monoisotopic, and heavy isotopic peaks led to the identification of 963 proteins (n ≥ 3; q ≤ 0.01; type II error ∼60%). Using the power of the OIT to resolve the Gaussian peaks from heavy isotopes and hydrogen loss resulted in the identification of three times more proteins with high confidence and a much lower type II error than analyzing peptides from the monoisotopic peak alone. The resolving power of the OIT may be exploited to increase observation frequencies and provide greater proteomic coverage and statistical power in comparative proteomics studies.
SDGs
Publisher
American Chemical Society (ACS)
Type
journal article