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J Proteome Res. 2017 Jul 7;16(7):2445-2456. doi: 10.1021/acs.jproteome.7b00061. Epub 2017 May 25.

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

Shen X1,2, Shen S3,2, Li J1,2, Hu Q4, Nie L2,5, Tu C1,2, Wang X4, Orsburn B6, Wang J4, Qu J1,2.

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Department of Pharmaceutical Science, SUNY at Buffalo , Buffalo, New York 14228, United States.
Center of Excellence in Bioinformatics & Life Sciences , Buffalo, New York 14203, United States.
Department of Biochemistry, SUNY at Buffalo , Buffalo, New York 14228, United States.
Roswell Park Cancer Institute , Buffalo, New York 14263, United States.
Shandong University , Shandong Sheng 250000, China.
ThermoFisher Scientific , Pittsburgh, Pennsylvania 15275, United States.


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.


LC−MS; MS1-based; Orbitrap; false-positive biomarker discovery rate; ion current; label-free; large cohorts; missing data; proteomics; quantitative proteomics

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