ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry

Abstract

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789.

Keywords: electron capture dissociation (ECD); internal fragment; terminal fragment; top-down mass spectrometry (TD-MS).

Figure 1.

A. The graphical user interface (GUI) for ClipsMS. The user can input several key parameters including the error allowed, the smallest internal fragment size, the sequence, the observed fragments, any modifications on the sequence and the type of fragments to search. B. The workflow of the algorithm and how it matches peaks input by the user. The algorithm calculates all theoretical terminal and internal fragments, matches all peaks, makes decisions on which assignments to keep, and automatically generates figures.

Figure 2.

A. Broadband ECD MS of 20 μM apo-myoglobin formed from acidic denaturing conditions. B. A fragment location map indicating the region of the protein sequence covered by terminal and internal fragments. C. A sequence coverage map for the terminal and internal fragments. Darker regions indicate more coverage. D. A fragment cleavage map indicating the location of inter-amino acid cleavage sites for terminal and internal fragments.

Figure 3.

A. Broadband ECD MS of 20 μM oxidized apo-myoglobin formed from acidic denaturing conditions. B. A fragment location map indicating the region of the protein sequence covered by terminal and internal fragments. Dashed lines indicate sites of oxidation. C. A sequence coverage map for the terminal and internal fragments assigned indicating terminal and internal fragments cover both oxidation sites. Darker regions indicate more coverage. D. A fragment cleavage map indicating the location of inter-amino acid cleavage sites for terminal and internal fragments. Red amino acids indicate sites of oxidation.