ExMS: data analysis for HX-MS experiments

Abstract

A previous paper considered the problems that presently limit the hydrogen exchange-mass spectrometry (HX-MS) method for studying the biophysical and functional properties of proteins. Many of these problems can be overcome by obtaining and analyzing hundreds of sequentially overlapping peptide fragments that cover the protein many times over (Mayne et al. J. Am. Soc. Mass Spectrom. 2011: 10.1007/s13361-011-0235-4). This paper describes a computer program called ExMS that furthers this advance by making it possible to efficiently process crowded mass spectra and definitively identify and characterize these many peptide fragments. ExMS automatically scans through high resolution MS data to find the individual isotopic peaks and isotopic envelopes of a list of peptides previously identified by MS/MS. It performs a number of tests to ensure correct identification in spite of peptide overlap in both chromatographic and mass spectrometric dimensions and possible multi-modal envelopes due to static or dynamic structural heterogeneity or HX EX1 behavior. The program can automatically process data from many sequential HX time points with no operator intervention at the rate of ~2 sec per peptide per HX time point using desktop computer equipment, but it also provides for rapid manual checking and decision when ambiguity exists. Additional subroutines can provide a step by step report of performance at each test along the way and parameter adjustment, deconvolute isotopic envelopes, and plot the time course of single and multi-modal H-D exchange. The program will be available on an open source basis at: http://HX2.med.upenn.edu/download.html.

Figure 1

Workflow summarizing the operational steps performed by the ExMS program

Figure 2

Illustration of ExMS processing for one peptide from an all-H sample. (A) TIC for the peptic peptides of an all-H sample. ExMS analyzes each scan within a settable window (yellow band) around the RT of each search peptide taken from a previous MS/MS analysis (dashed red line at 3.95 min for peptide 4–17 +3). (B) The m/z spectrum of one such scan (at RT=3.81 min). (C) Expanded section from (B). ExMS finds peaks in each scan within the RT search range (9 point method, peaks shown). They are compared with the theoretical m/z of the search peptide peaks (dashed red lines). Potentially matching peaks (theoretical±10 ppm) and their envelope are tested to ensure a correct match. Result of the intensity distribution test is shown (isotopic envelope compared with theoretical intensity distribution). Scans that pass all tests are marked as matched. Matched spectra are summed and the summed spectrum is subjected to the same tests plus others (autocheck stage) to accept or question the assignment. (D) For each scan in the search range, a plot of the summed intensity of each matched envelope (I, black) and its score in the intensity distribution test (R², red). The I×R² parameter and other tests are used to specify the all-H RT range for that peptide for that day (yellow band). For subsequent analysis of deuterated samples, scans in this narrow RT range are analyzed similarly to identify each listed peptide, and measure its centroid mass, and therefore the number of carried deuterons

Figure 3

Peak matching example shown for two closely overlapping peptides. Comparison of experimental and theoretical peak positions and envelopes for peptides 61–73 +3 and 77–90 +3. The ExMS analysis finds each peptide in its turn, automatically, in the autocheck stage. In this case, both pass all tests. This figure illustrates the ability of ExMS to distinguish spectrally overlapping envelopes from different peptides, even when the isotopic peaks are nearly isobaric (inset) and the envelopes are bimodal. These data, taken from an H-D exchange pulse labeling experiment during kinetic protein folding, exhibit a transient folding intermediate and some already folded native protein. Additional data taken as a function of pre-folding time (not shown) show the rate for formation and loss of the intermediate and for formation of the native protein

Figure 4

Intensity out of range tests. (A) Expected m/z positions of isotopic peaks for the search peptide, relative to m_mono/z taken as zero, are marked in red. Locations marked in black are examined to check for a possible misassignment due to a co-eluting and overlapping peptide (check at (m_mono –1)/z and at (m_mono+maxH+maxD+1)/z. Locations marked in blue are examined to check for a possible misassignment due to a peptide with higher charge state indicated by intervening peaks. ExMS checks up to z=8; z=2 to 6 are shown; actual positions will depend on the charge state of the search peptide. (B) In the search for peptide 14–39 +4, ExMS finds the possible match shown but this identification is questionable due to the presence of a significant out of range peak at (m_mono –1)/z (black x). The scan is therefore marked not matched. (C) In the search for peptide 76–92 +2, ExMS finds the possible match shown, but this peptide fails the out of range test due to intervening peaks (blue). The scan is therefore marked not matched

Figure 5

Intensity distribution test. Theoretical isotopic peak positions are in red. (A) The match between theoretical and experimental peak intensities for an all-H sample. (B) Bi-Gaussian fit to a fully deuterated sample. (C) Bi-Gaussian fit to a partially deuterated sample. All three potential identifications pass the intensity distribution test since R² is >0.7. (These data, from a time-dependent H-D exchange experiment on α-synuclein amyloid (Parkinson’s disease), demonstrate static structural heterogeneity near the molecular N-terminus, define its position, and measure the population fractions. Time-dependent HX data (not shown) measures the segment-resolved stability of each)

Figure 6

Sample ExMS manual check presentation. Two different manual check presentations are shown. The search for 61–75 +2 (left) in a deuterated sample does not pass the autocheck because ExMS finds an isobaric peptide in the peptide list (62–76 +2), and the converse in the right windows. The upper window shows results on the retention time axis including (top panel) the RT value for the search peptide (fromMS/MS; red dashed line), the RT range determined by ExMSfor this peptide in the all-H sample (MS; yellow band, as in Figure 2), and the RT range for the two suggested peptide envelopes in the deuterated sample (MS; horizontal red lines). The next two panels in this window assess the quality of the possible match. As in Figure 2D, they show plots of the summed intensity of the isotopic peaks of the peptide spectrum in each individual scan (SEIC is summed extracted ion current), and the intensity distribution test R² value of each scan. The lower window (C) and (D) shows MS data for the putative search peptide, compared to expected m/z locations of the search peptide (red) and the confusing isobaric peptide (blue). The red and blue m/z positions are identical because the peptides in question are isobaric. The lower panel shows the fitted spectrumputatively found for the search peptide (intensity distribution test for the summed scans; bi-Gaussian fit to the MS data), and also demonstrates the absence of other ambiguous peaks (intensity out of range test). Given this display, the operator can quickly judge that the results definitively remove the initial ambiguity and clearly identify the MS spectra of the two isobaric peptides. Data are from the same H-D exchange pulse labeling experiments in Figure 3 showing a folding intermediate and some already folded protein. Here the peptides shown are absolutely isobaric as well as spectrally overlapping, but they can be individually identified in the manual check stage because they separate chromatographically (top panel)