Improved strategies for rapid identification of chemically cross-linked peptides using protein interaction reporter technology

Abstract

Protein interaction reporter (PIR) technology can enable identification of in vivo protein interactions with the use of specialized chemical cross-linkers, liquid chromatography, and high-resolution mass spectrometry. PIR-cross-linkers contain labile bonds that are specifically fragmented under low energy collision or photodissociation conditions in the mass spectrometer source, thus releasing cross-linked peptides. Successful analysis of PIR-cross-linked proteins requires the use of expected mathematical relationships between cross-linked complexes and released peptides after fragmentation of the labile PIR bonds. Presented here is a next-generation software tool, BLinks, for use in the analysis and identification of PIR-cross-linked proteins. BLinks is an advancement beyond our previous efforts by incorporation of chromatographic profiles that must match between cross-linked complexes and released peptides to enable estimation of p-values to help filter true relationships from complex data sets. Additionally, BLinks was used to incorporate Mascot database searching results from subsequent MS/MS analysis of the released peptides to facilitate identification of cross-linked proteins. BLinks was used in the analysis of human serum albumin, and 46 interpeptide relationships were found spanning 30 proximal residues with a 2.2% false discovery rate. BLinks was also used to track peptides involved in multiple, coeluting relationships that make accurate identification of protein interactions difficult. An additional 10 interpeptide relationships were identified despite poor correlation using the profiling tools provided with BLinks. Additionally, BLinks can be used to globally map all interpeptide relationships from the data analysis and customize subsequent analysis to target specific peptides of interest, thus making it a useful tool for both discovery of protein interactions and mapping protein topology.

Figure 1

Illustration of PIR cross-linking technology. (A) The chemical structure for the in-house synthesized cross-linker, Brink. Shown in (B) is a cartoon illustration of Brink, highlighting the affinity group and mass encoded tag, the labile bond regions, and the reactive groups. (C) Three general PIR products are formed from the fragmentation of PIR-linked peptides: dead-end, intra-cross-links, and inter-cross-links. Dead-ends and intra-cross-linked relationships are made from the contribution of a single peptide mass and the reporter ion mass. Inter-cross-linked relationships involve two peptide ions and the reporter ion.

Figure 2

Illustration of PIR fragmentation and data acquisition using in-source collision induced dissociation (ISCID). Use of ISCID is alternated between each spectrum acquisition, generating mass spectra with either intact PIR precursor ions, or fragmented PIR product ions. Inter-cross-linked relationships are made from the summation two peptide ion masses and the reporter ion mass in the product ion scans to produce the intact precursor ion mass observed in the previous scan event.

Figure 3

Extracted ion chromatogram correlation for inter-crosslinked peptides. The chromatogram intensities shared between the intact PIR-linked ion and the two short arms (indicated in the blue boxes of A) are used to produce three correlation scores (B) relating 1) the intact PIR-linked ion to the first short arm, 2) the intact PIR-linked ion to the second short arm, and 3) the two short arms to each other.

Figure 4

Histogram of the distances between ε-amines of inter-cross-linked peptides. The distances were calculated from the crystal structure of HSA.

Figure 5

(A) The correlation graph showing poor correlation between the intact PIR-linked ion and the second short arm in the relationship. (B) The relationship map for the second short arm shows that it is involved in two inter-crosslinked relationships, with an ion of mass 614.38 Da and an ion of mass 1725.76 Da. (C) The extracted ion chromatograms for the intact PIR-linked ion and the second short arm. The blue boxes in (B) and (C) indicate the region over which the correlation in (A) is made for the relationship. The contribution of the ions from the second inter-crosslinked peptide relationship are the cause of the poor correlation.

Figure 6

Profile of all PIR relationships identified with BLinks. Inter-cross-links form a distinct cluster from dead-ends and intra-cross-links when plotted by retention time and mass.

Figure 7

Complete PIR relationship profiles for two inter-cross-linked peptides. The specific peptides involved in a single inter-cross-linked relationship are highlighted to show their involvement in other cross-linked relationships.