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.

Figure 1

FAIMS effect on PRM analysis of EGFR IPLENLQIIR [M + 2H]²⁺. (A) CV scan for EGFR IPLENLQIIR [M + 2H]²⁺ with direct infusion of synthetic peptide, using FAIMS CVs from – 100 to 0 V, step size 1. The plot shows the MS/MS intensity of the precursor (604.87 m/z) with HCD collision energy set to 0. CVs corresponding to half-maximal intensity are labeled. (B) Fragmentation map of IPLENLQIIR. (C, D) MS/MS spectrum at the apex of 46-amol EGFR IPLENLQIIR elution spiked into trypsin-digested, formalin-fixed spleen (C) with the optimized FAIMS CV and (D) without FAIMS. Fragment ions derived from EGFR IPLENLQIIR are labeled and colored. Ion count refers to total ion count in the Orbitrap cell, recorded as RawOvFtT in the scan header. (E, F) XICs of PRM fragment ions of IPLENLQIIR [M + 2H]²⁺ at 46 amol/μg with (E) FAIMS-PRM and (F) PRM.

Figure 2

Peptides with a FAIMS-dependent improvement in LLOQ also have a greater reduction in background signal. (A) Background ions per scan, with and without FAIMS for elution time and m/z regions of peptide targets. (B) The percentage reduction in background signal with FAIMS is shown, color-coded by reduction in LLOQ with FAIMS. Background ions are calculated for blank injections and heavy m/z windows only, with the XIC window determined from the light reference peptide elution (n = 8, from n = 2 LOQ curves per condition; number of scans per plot ranges from 360 to 948). Ion numbers are from ‘RawOvFtT’, extracted from raw files using rawDiag (Trachsel et al., J Proteome Research 2018). Peptides are annotated with precursor sequence and charge-state. Labels on the lower panel indicate the percentage reduction in background signal for each target.

Figure 3

(A) Workflow of clinical proteomics with FAIMS-PRM. (B) Quantitation of EGFR IPLENLQIIR [M + 2H]²⁺ from two breast cancer samples. The upper XICs are from endogenous signals, and the lower XICs are from the heavy peptide reference signal (5 fmol). The LLOQ with FAIMS for the y8 fragment ion of this peptide was 46 amol/µg. (C) Quantitation of HER2 ELVSEFSR [M + 2H]²⁺ from two breast cancer samples. The upper XICs are from endogenous signal, and the lower XICs are corresponding heavy-peptide reference signals (5 fmol). Inset shows magnified view of the fragment ions for the 210-amol/µg sample. The LLOQ with FAIMS for the y5 fragment ion of this peptide was 46 amol/µg.

Figure 4

Quantitation of HER2 by FAIMS-PRM in 20 breast cancer biopsies. (A) Scatterplot showing the correlation between HER2 ELVSEFSR and HER2 SGGGDLTLGLEPSEEEAPR peptides. The Pearson correlation coefficient squared value is shown; inset shows expanded view of region below 800 amol/µg. (B) HER2 concentrations by FAIMS-PRM for the ELVSEFSR peptide are shown for each of the HER2 classifications by IHC. All MS values were above the LLOQ.