High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer

Abstract

The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.

Keywords: HeLa; Orbitrap mass spectrometer; higher-energy collisional dissociation; protein; proteoform; proteomics; top-down; ultraviolet photodissociation.

Figure 1

Proteins (left) and proteoforms (right) detected by high-throughput top-down proteomic analysis of seven HeLa GELFrEE fractions using HCD or UVPD fragmentation.

Figure 2

(A) -log(q-value), (B) molecular weight, and (C) C-score distributions for all detected proteoforms within the HCD (dark green) and UVPD (light green) datasets.

Figure 3

(A) -log(q-value) and (B) molecular weight distributions of the 286 overlapping proteoforms between the HCD (dark green) and UVPD (light green) datasets.

Figure 4

Distribution of gains in UVPD compared to HCD for (A) C-scores and (B) sequence coverage for characterization of proteoforms. Overlays of normal distributions and kernel distributions are given for each metric.

Figure 5

Fragmentation maps of A) EDRK-rich factor 2 (PFR21392) for HCD (12+) and UVPD (12+), and B) high mobility group protein HMG-I with three post-translational modifications (PFR17157) for HCD (14+) and UVPD (17+). Specific residues or sites are shaded as follows: blue box = phosphorylation, red box = acetylation, green box = methylation. Backbone fragmentation markers are shown along the sequence as colored flags: a,x: green; b,y: blue; c,z: red.

Figure 6

Fragmentation maps of A) heat shock protein beta-1 (PFR20705) for HCD (29+) and UVPD (26+), and B) ATP synthase subunit g (mitochondrial) (PFR16756) for HCD (12+) and UVPD (12+). UVPD allows localization of the acetylation (red box) of lysine-10, but HCD does not. Specific residues or sites are shaded as follows: blue box = phosphorylation, red box = acetylation, green box = methylation. Backbone fragmentation markers are shown along the sequence as colored flags: a,x: green; b,y: blue; c,z: red.Specific residues or sites are shaded as follows: blue box = phosphorylation, red box = acetylation, green box = methylation. Backbone fragmentation markers are shown along the sequence as colored flags: a,x: green; b,y: blue; c,z: red.