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.

High-Sensitivity Detection toward SARS-CoV-2 S1 Glycoprotein by Parallel Reaction Monitoring Mass Spectrometry

“…Second, the separation gradient should be condensed to shorten the analysis time. Furthermore, high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with a PRM assay could separate the gas phase ions online to reduce the chemical background in the MS, which could further enhance the detection sensitivity . Ultimately, the MS-based targeted PRM assay is expected to achieve large-scale clinical applications as a routine diagnostic tool.…”

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: Practical Alternative for Cardiac Proteome Sample Processing

“…Combining multiple compensation voltages (CVs) within a single run or between runs improves whole proteome coverage in cell lysates. , For example, Pfammatter et al observed a 30% gain in unique peptide identification using FAIMS compared with non-FAIMS analysis of HEK 293 cells . Other studies using HeLa protein digest such as Johnson KR et al, Greguš M et al, and Cong Y et al concurrently showed the addition of FAIMS had significantly increased protein and peptide identifications than without FAIMS. − In a few recent studies working with various tissue samples, including brain autopsies, tumor biopsies, and paraffin embedded tissues (lymph node, lung, and prostate), the addition of FAIMS also showed an enhanced detection of nonredundant 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 with previous established fractionation methods.…”