The addition of FAIMS Increases Targeted Proteomics Sensitivity from FFPE Tumor Biopsies

Abstract

Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM. Limits of quantitation were determined by spiking synthetic peptides into a human spleen matrix. In addition, 20 clinical samples were analyzed using FAIMS-PRM and the quantitation of HER2 was compared with that obtained with the Ventana immunohistochemistry assay. FAIMS-PRM improved the overall signal-to-noise ratio over that from PRM and increased assay sensitivity in FFPE tissue analysis for four (HER2, EGFR, cMET, and KRAS) of five proteins of clinical interest. FAIMS-PRM enabled sensitive quantitation of basal HER2 expression in breast cancer samples classified as HER2 negative by immunohistochemistry. Furthermore, we determined the degree of FAIMS-dependent background reduction and showed that this correlated with an improved lower limit of quantitation with FAIMS. FAIMS-PRM is anticipated to benefit clinical trials in which multiple biomarker questions must be addressed and the availability of tumor biopsy samples is limited.

Don't let go: co‐fractionation mass spectrometry for untargeted mapping of protein–metabolite interactomes

“…By changing the compensation voltage over time, different groups of ions are selected, which results in increased proteome coverage over FAIMS‐free strategies. The combination of FAIMS and MS offers significant benefits, as it: (i) increases proteome coverage and reduces the extent of peptide co‐fragmentation (Hebert et al., 2018; Pfammatter et al., 2018); (ii) increases the sensitivity of the targeted acquisition methods (Sweet et al., 2022); (iii) increases the identification rate of phosphopeptides and facilitates the resolution of their isomeric variants (Muehlbauer et al., 2020); (iv) enriches the highly charged cross‐linked peptides (Steigenberger et al., 2020); and (v) depletes the highly abundant peptides used for protein elution in IP experiments (Ang et al., 2022).…”

High Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry to Enhance Cardiac Muscle Proteome Coverage

“…[29], concurrently showed FAIMS had significantly increased in protein and peptide identifications than without FAIMS. In a few recent studies working with various tissue samples, including brain autopsies [30], tumor biopsies [31], or paraffin embedded tissues (lymph node, lung, and prostate) [32], the addition of FAIMS also showed enhanced detection of non-redundant proteoforms, increased proteome sensitivity, or substantially reduced sample handling. As the cardiac proteome consists of large dynamic range, it remains unclear whether FAIMS can detect peptides and the less abundant proteins from the cardiac proteome that is comparable to the previous established fractionation methods.…”