# Comparison of the Human Plasma Peptides from the Fit of Fragmentation Spectra versus Accurate Monoisotopic Precursor Mass

## TL;DR

This study compares two mass spectrometry techniques for identifying plasma peptides by analyzing fragmentation spectra and precursor mass accuracy.

## Contribution

The study demonstrates that accurate peptide identification in human plasma can be achieved using MS/MS spectra alone without precise precursor mass measurements.

## Key findings

- Peptide identifications using MS/MS spectra alone showed high agreement between OIT and LIT instruments.
- Delta mass plots matched predicted isotope and hydrogen rearrangement distributions, validating MS/MS-based identification.
- Over 99.9% overlap in protein gene symbols identified by both instruments confirmed robust and reproducible results.

## Abstract

In nature, ionized peptides with heavy isotopes and hydrogen
rearrangements
show a broad mass distribution with signals at discrete delta mass
values from −3 to +5 Da by mass spectrometry (MS). For many
peptides, the intensity of the +1 or +2 Da isotope exceeds the signal
from the monoisotopic mass. Therefore, there is a need for a method
that improves peptide identification from heavy isotopes or hydrogen
rearrangements based on the fit of tandem mass spectra. Peptides may
be identified using an accurate monoisotopic precursor mass with ≤0.1
Da. However, many peptides with heavy isotopes and H-loss can be identified
and enumerated based on the fit of their MS/MS spectra alone in the
absence of an accurate precursor monoisotopic mass (i.e., ± 3
Da) using the X!TANDEM MS/MS fitting algorithm. In this study, human
plasma samples were analyzed with a highly resolving axially harmonic
orbital ion trap (OIT) and a sensitive linear quadrupole ion trap
(LIT). The MS/MS fragmentation spectra from the OIT can be fit to
peptides from the monoisotopic (±0.1 Da) as well as all other
precursor masses with a wide mass tolerance (±3 Da). The resulting
delta mass distribution can then be plotted and compared to the predicted
distribution of heavy isotopes and hydrogen rearrangements to provide
a direct biophysical prediction and test the validity of the fit determined
by accepting the best-fit MS/MS spectra. The OIT instrument, which
has greater resolution, was sampled at 30 nL per minute, while the
more sensitive LIT was sampled at 200 nL per minute. The MS/MS spectra
generated by each instrument were fit to peptides within a wide window
(±3 Da) using the rigorous X!TANDEM algorithm. The OIT and LIT
results were compared in an SQL Server database and corrected against
analytical and statistical controls. The delta mass distribution of
the peptides with hydrogen rearrangements and heavy isotopes was determined
from the fit MS/MS spectra using the R statistical program. The OIT
sampled MS and MS/MS spectra from the high-intensity precursor ions
by focusing on E7 to E9 detector counts. In contrast, the LIT sampled
a range of precursor ion intensities focused from E4 to E7 and thus
reached lower ion intensity values. As expected, the precursor mass
[M + H]+ obtained by the OIT exhibited sharp delta mass
peaks at −3, −2, −1, 0, +1, +2, +3, +4, and +5
Da due to naturally occurring heavy isotopes and hydrogen rearrangements.
The collection of peptides and proteins identified by OIT and LIT
was in qualitative and quantitative agreement with one another, with
99.9% overlap on 2726 protein gene symbols from human plasma and a
highly significant relationship by regression analysis. The protein p-values, false discovery rate q-values,
and comparisons to the noise MS/MS analytical control and random MS/MS
statistical control confirmed the high-confidence MS/MS identifications
from both instruments. MS/MS fragmentation spectra from the OIT were
fit to peptides. The resulting precursor ion delta mass distribution
showed a precise match to the predicted isotope distributions and
hydrogen rearrangements of natural peptides. Thus, analysis of delta
mass plots provided powerful biophysical evidence for the accuracy
of plasma peptide identification from the fit of the MS/MS spectra
alone. The high level of agreement on proteins and peptides and the
proportional enumeration between proteins identified by the OIT and
those identified independently using a LIT confirmed that plasma peptides
and proteins may be identified and quantified from MS/MS spectra alone
without the need for an accurate measure of the precursor mass. The
greater sensitivity and low cost of searching MS/MS spectra in the
absence of an accurate mass mean that it is possible to identify and
quantify more proteins for the discovery of proteins in clinical populations.