Evaluation of Disposable Trap Column nanoLC–FAIMS–MS/MS for the Proteomic Analysis of FFPE Tissue

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 a high proteomic depth and limiting the
overall analysis time. To this end, we evaluated the merits of online
coupling of single-use disposable trap column nanoflow liquid chromatography,
high-field asymmetric-waveform ion-mobility spectrometry (FAIMS),
and tandem mass spectrometry (nLC–FAIMS–MS/MS). The
data show 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 (CVs) as a function of the employed LC gradient time. We
found nLC–FAIMS–MS/MS to be on par with StageTip-based
off-line basic 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 materials
from the lymph node, lung, and prostate tissue as examples, we show
that nLC–FAIMS–MS/MS can identify 5000–6000 proteins
from the respective tissue within a total of 3 h of analysis time.