Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down Proteomics

Abstract

Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.

Figure 1.

Total ion chromatograms (TICs) of top-down standards using either FAIMS at different CVs or no FAIMS. Top-down standards run without FAIMS (top panel) and with FAIMS using compensation voltages (CV) of −30 V, −20 V, −10 V, 0 V, + 10 V, and +20 V (lower panels). Top-down standard proteins: ubiquitin, 8.6 kDa (U); trypsinogen, 24 kDa (T); myoglobin, 16.7 kDa (M); and carbonic anhydrase, 29 kDa (C).

Figure 2.

Protein and proteoform identifications with or without FAIMS. (A) Histogram of CD3⁺ T cell proteoforms identified at each compensation voltage (CV). (B, C) Venn diagrams of proteins (panel (B)) and proteoforms (panel (C)) identified from CD3⁺ T cells subjected to top-down proteomics analysis with FAIMS or without FAIMS.

Figure 3.

Changing the compensation voltage (CV) results in detection of a hidden population of proteins through improved signal-to-noise values. (A) Protein-level heatmap of CD3⁺ T cell proteins identified without FAIMS (8 technical replicates) or with FAIMS applying different CVs from −40 V to +30 V. CVs for injections analyzed with FAIMS are indicated at the bottom of the heatmap. (B) Mass spectrum of proteoform PFR295102 from UniProt accession P20292 (Arachidonate 5-lipoxygenase-activating protein) analyzed with FAIMS (top panel) and without FAIMS (lower panel) at same retention time. (C) Fragmentation map of PFR295102 showing 52% sequence coverage in the sample using FAIMS.