MassShiftFinder

Blind search for post-translational modifications and amino acid substitutions using peptide mass fingerprints from two proteases.

Introduction

The identification of post-translational modifications and amino acid substitutions caused by mutations or nucleotide polymorphisms is an essential part of protein characterization. The most common way of doing characterization is by mass spectrometry, where the protein is digested by a protease creating a peptide mass fingerprint (PMF). There are two main approaches: (i) using a predefined set of possible modifications and substitutions or (ii) performing a blind search. The first option is straightforward, but detecting modifications or substitutions outside of the predefined set is not possible. Blind search does not have this constraint, and therefore has the potential of detecting both known and unknown modifications and substitutions. Combining the peptide mass fingerprints from two proteases result in overlapping sequence coverage of the protein, thereby offering alternative views of the protein and a novel way of indicating post-translational modifications and amino acid substitutions.

MassShiftFinder is a software tool for doing blind search using peptide mass fingerprints from two proteases with different cleavage specificities. The algorithm relies on overlapping peptides for the two proteases used, and can indicate both modifications and amino acid substitutions. The method can help restrict the area where the modification has occurred.

Example

Consider the protein sequence …L|GHVVSTPTK/AL|QDNK/... where / indicates a tryptic cleavage and | indicates a chymotryptic cleavage. With a complete cleavage, the tryptic and chymotryptic peptides overlap at the two residues, AL. Assume that the A has become an S due to a polymorphism. The corresponding tryptic and chymotryptic peptides then get a mass approximately 15.995 Da higher than expected, referred to as a mass shift, and therefore do not result in a match with the theoretical peptides. A general search for substitutions could be done for each of the proteases separately, but this would result in several false positives (in the present sequence A to S, S to C, P to L/I, V to D, L to E and D to M all give mass shift of 16). The combination of data from two proteases can increase the confidence and restrict the choices of possible modifications/substitutions, as this results in two indicators that a substitution or a modification has occurred. Furthermore, it is possible to restrict the area where the change is positioned, and the relevant peptides can then be targeted experimentally, i.e., by MALDI-TOF-TOF.

Overlapping peptides

The concept of overlapping peptides is formalized in Figure 1. Assume two overlapping peptides, p1 and p2, generated by two different proteases. Let p1 be the most N-terminal peptide, and p2 the most C-terminal peptide. As a direct consequence of the overlapping, three areas are defined: the overlapping area Y; the area of p1 not overlapping with p2 X (the most N-terminal area); and the area of p2 not overlapping with p1 Z (the most C-terminal area). Together these areas are called the covered area. Note that if p1 and p2 have the same start or end residue, the areas X or Z will contain no residues (typically, the N- or C-terminus of the protein). Also note that one of the peptides can be completely covered by the other. The main idea of the algorithm is that a modification or amino acid substitution occurring in the area Y will be detected as an equal mass shift in p1 and p2.

Figure 1: The overlapping areas between the two peptides p₁ and p₂.

Equal mass shifts occurring in X and Z, but not in Y, will also be detected. This means that the non-overlapping areas X and Z both contain the same amino acid substitution or the same modified amino acid, or different amino acids carrying an identical modification, e.g., phosphorylation on S and T. Other peptides providing an alternative coverage of the area Y, e.g., due to missed cleavages in p5 (= p1 + p3) or p6 (= p2 + p4), and/or an alternative coverage of the areas X and/or Z (peptides p3, p4, p5 and p6), may improve the confidence in a mass shift and its location.