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Evaluation of disposable-trap column nanoLC-FAIMS-MS/MS for the proteomic analysis of FFPE tissue

ABSTRACT: Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving high proteomic depth and limiting overall analysis time. To this end, we evaluated the merits of online coupling of disposable trap column nano-flow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data shows that ≤ 600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CV) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with stage tip-based off-line high pH reversed phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤ 15% for 90 % of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE material from lymph node, lung and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5-6,000 proteins from the respective tissue within a total of 3 hours of analysis time.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Prostate Gland, Lung, Permanent Cell Line Cell, Lymph Node

SUBMITTER: Stephan Eckert

LAB HEAD: Bernhard Kuster

PROVIDER: PXD028136 | Pride | 2021-11-02

REPOSITORIES: Pride

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