Selected Ion Extraction of Peptides with Heavy Isotopes and Hydrogen Loss Reduces the Type II Error in Plasma Proteomics

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.