Many overlapping peptides for protein hydrogen exchange experiments by the fragment separation-mass spectrometry method

Abstract

Measurement of the naturally occurring hydrogen exchange (HX) behavior of proteins can in principle provide highly resolved thermodynamic and kinetic information on protein structure, dynamics, and interactions. The HX fragment separation-mass spectrometry method (HX-MS) is able to measure hydrogen exchange in biologically important protein systems that are not accessible to NMR methods. In order to achieve high structural resolution in HX-MS experiments, it will be necessary to obtain many sequentially overlapping peptide fragments and be able to identify and analyze them efficiently and accurately by mass spectrometry. This paper describes operations which, when applied to four different proteins ranging in size from 140 to 908 residues, routinely provides hundreds of useful unique peptides, covering the entire protein length many times over. Coverage in terms of the average number of peptide fragments that span each amino acid exceeds 10. The ability to achieve these results required the integrated application of experimental methods that are described here and a computer analysis program, called ExMS, described in a following paper.

Figure 1

On-line HX-MS analysis system. The entire flow system is contained within a Peltier cooled chamber (21× 15×25 cm; from an automobile accessory supplier) that maintains 0±1 °C, monitored with a thermocouple thermometer. An internal fan circulates air across the Peltier element and through the chamber. Liquid flow is precooled in a large loop positioned in the airflow. Switching valves are mounted on a ridged board backed with foam insulation with valve handles outside easily accessible for manual operation. The wetted parts of the valves, the injection loop, and columns are contained in the cold chamber. Liquid lines and the thermometer leads are threaded through small holes in the insulation and mounting board. A short length of outflow tubing from the C18 analytical column to the MS electrospray source is packed with ice-filled plastic bags

Figure 2

Distributions of Bioworks P_pep scores. Known false identifications, in red, represent SEQUEST hits for MS/MS data run against a large database with reversed sequences. Data in blue represent hits on the experimental protein for MS/MS data run against the large database with all proteins in their forward sequences. Results for the four proteins studied are merged to provide a large statistical sampling. The inset focuses on the poorest scoring matches. These results show that the cutoff P_pep value necessary to reduce false identifications to <0.1% is 0.990. (Other measures of assignment quality could be calibrated in the same way.)

Figure 3

Unique peptides and amino acid coverage obtained for four proteins by the methods described here. Peptides are represented by horizontal bars. The different colors distinguish peptides obtained by proteolysis with porcine pepsin (red), fungal protease XIII (blue), and the two proteases in tandem (green). The coverage graphs show the number of peptides that span each amino acid residue position. For each protein, the list of peptides identified by MS/MS was culled to reduce potentially false identifications to one per thousand, and to eliminate those not definitively found by the ExMS program in MS spectra from runs with all-H protein and 50% D-labeled protein