An IonStar Experimental Strategy for MS1 Ion Current-Based Quantification Using Ultrahigh-Field Orbitrap: Reproducible, In-Depth, and Accurate Protein Measurement in Large Cohorts

In-depth
and reproducible protein measurement in many biological
samples is often critical for pharmaceutical/biomedical proteomics
but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field
Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically
important experimental approaches enabling reliable large-cohort analysis
have long been overlooked. Here we described an IonStar experimental
strategy achieving excellent quantitative quality of MS1 quantification.
Key features include: (i) an optimized, surfactant-aided sample preparation
approach provides highly efficient (>75% recovery) and reproducible
(<15% CV) peptide recovery across large cell/tissue cohorts; (ii)
a long column with modest gradient length (2.5 h) yields the optimal
balance of depth/throughput on a Q/UHF-Orbitrap; (iii) a large-ID
trap not only enables highly reproducible gradient delivery as for
the first time observed via real-time conductivity monitoring, but
also increases quantitative loading capacity by >8-fold and quantified
>25% more proteins; (iv) an optimized HCD-OT markedly outperforms
HCD-IT when analyzing large cohorts with high loading amounts; (v)
selective removal of hydrophobic/hydrophilic matrix components using
a novel selective trapping/delivery approach enables reproducible,
robust LC–MS analysis of >100 biological samples in a single
set, eliminating batch effect; (vi) MS1 acquired at higher resolution
(fwhm = 120 k) provides enhanced S/N and quantitative accuracy/precision
for low-abundance species. We examined this pipeline by analyzing
a 5 group, 20 samples biological benchmark sample set, and quantified
6273 unique proteins (≥2 peptides/protein) under stringent
cutoffs without fractionation, 6234 (>99.4%) without missing data
in any of the 20 samples. The strategy achieved high quantitative
accuracy (3–6% media error), low intragroup variation (6–9%
media intragroup CV) and low false-positive biomarker discovery rates
(3–8%) across the five groups, with quantified protein abundances
spanning >6.5 orders of magnitude. Finally, this strategy is straightforward,
robust, and broadly applicable in pharmaceutical/biomedical investigations.