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.

Desalted samples (1.2 µg) were combined with synthetic isotope-labelled peptides (6 fmol). Five-sixths of this mixture (1 µg of total peptide and 5 fmol of each synthetic peptide) was loaded onto EvoTip trapping columns before separation with the EvoSep One nanoLC system (EvoSep, Odense, Denmark) coupled to an Orbitrap Fusion Lumos mass spectrometer with a FAIMS-PRO interface (Thermo Fisher). Peptides were eluted over a 44-min gradient, from 7% to 30% acetonitrile (on-column), at a flow rate of 500 nL/min (Supplemental Materials and Methods). The FAIMS-PRM experiment employed higher-energy collisional dissociation (HCD) fragmentation with an isolation window of 0.7 mass-to-charge ratio (m/z), a target automatic gain control of 1E6 ions, and a maximum injection time of 100 ms. Tandem MS (MS/MS) scans were acquired in centroid mode with the Orbitrap detector, using 30K resolution at 200 m/z. FAIMS was operated at the standard resolution, with no additional FAIMS gas.

LOQ curve samples: Bulk formalin-fixed human spleen, homogenized and prepared as described above, was used as a complex matrix to determine the limit of quantitation (LOQ). A reverse-dilution LOQ experiment was carried out with spiking of constant light synthetic peptide (5 fmol) and a dilution series of heavy stable-isotope–labeled synthetic peptide. A 10-point dilution series was acquired in quadruplicate, ranging from 5 amol to 100 fmol on-column. Additional single and double blanks (reference peptide plus matrix; matrix only) were also acquired in quadruplicate Clinical samples: FFPE tumor biopsy samples from breast cancer patients, collected under Institutional Review Board approval by certified medical pathologists, were purchased from ProteoGenex (Inglewood, CA). Three tissue sections per sample were generated for hematoxylin and eosin (H&E) staining, HER2 IHC, and laser microdissection (LMD). Image analysis and LMD Pathology evaluation for laser-microdissected tumor epithelium was conducted by the study pathologist on H&E-stained slides, which were digitally imaged with an Aperio ScanScope AT scanner (Leica Microsystems, Wetzlar, Germany). HALO AI (Indica Labs, Albuquerque, NM) was used to classify and annotate the slides to guide LMD of tumor epithelium.