Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer - PubMed (original) (raw)

Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer

Lars Kolbowski et al. Anal Chem. 2017.

Abstract

We compared the five different ways of fragmentation available on a tribrid mass spectrometer and optimized their collision energies with regard to optimal sequence coverage of cross-linked peptides. We created a library of bis(sulfosuccinimidyl)suberate (BS3/DSS) cross-linked precursors, derived from the tryptic digests of three model proteins (Human Serum Albumin, creatine kinase, and myoglobin). This enabled in-depth targeted analysis of the fragmentation behavior of 1065 cross-linked precursors using the five fragmentation techniques: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), and the combinations ETciD and EThcD. EThcD gave the best sequence coverage for cross-linked m/z species with high charge density, while HCD was optimal for all others. We tested the resulting data-dependent decision tree against collision energy-optimized single methods on two samples of differing complexity (a mix of eight proteins and a highly complex ribosomal cellular fraction). For the high complexity sample the decision tree gave the highest number of identified cross-linked peptide pairs passing a 5% false discovery rate (on average ∼21% more than the second best, HCD). For the medium complexity sample, the higher speed of HCD proved decisive. Currently, acquisition speed plays an important role in allowing the detection of cross-linked peptides against the background of linear peptides. Enrichment of cross-linked peptides will reduce this role and favor methods that provide spectra of higher quality. Data are available via ProteomeXchange with identifier PXD006131.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1

Figure 1

(A) Workflow for inclusion list generation. Each protein was cross-linked and analyzed separately. (B) Evaluation of the identified cross-links against their corresponding protein crystal structures. The blue distribution reflects the distance between identified cross- link residue pairs. The gray distribution reflects all pairwise combinations of cross-linkable residues. The black line at 30 Å signifies the distance cutoff that was used to minimize false positives in the inclusion list. (C) Scheme of targeted analysis of cross-linked m/z species. (D) Reproducibility between the injection replicas with shuffled parameter order of the targeted cross-link experiments. Error bars representing the 0.95 confidence interval (CI) indicate reproducibility between the three different proteins.

Figure 2

Figure 2

Comparison of median number of MS2 spectra per second of different fragmentation methods when recording in the Orbitrap (Fusion Lumos). Percentages indicate loss in number of spectra compared to HCD.

Figure 3

Figure 3

Performance of fragmentation techniques depending on precursor m/z and charge. (A–E) Normalized sequence coverage of the best performing NCE for each method and m/z window. Numbers next to the mean values of the normalized sequence coverage indicate the respective best-performing NCE values in %. Error bars represent the 0.95 CI. (F) Resulting DDDT using the best fragmentation parameters from (A)–(E), for NCE consult the respective panels.

Figure 4

Figure 4

Performance of the NCE-optimized fragmentation methods for the pseudocomplex sample. (A) Mean numbers of identified cross-linked peptide pairs passing a 5% peptide pair level FDR over different search database sizes (error bars represent the 0.95 CI). (B) Total number of acquired MS2 spectra (averaged over three replica, dots show individual values). The stacked bars in the BSC bar indicate the number of spectra that were acquired with HCD (yellow, 90.5%) or EThcD (green, 9.5%). (C) Achieved sequence coverage comparison. Data refers to the PSMs from the cross-linked intraprotein pseudocomplex peptide pairs passing the 5% peptide pair FDR. (D) Performance comparison of the best method combinations for up to three injections of the same sample. Mean numbers of identified cross-linked peptide pairs passing a 5% peptide pair level FDR (error bars represent the 0.95 CI). Database size is eight sequences.

Figure 5

Figure 5

Performance of the NCE-optimized fragmentation methods for the ribosomal sample. (A) Mean numbers of identified cross-linked residue pairs passing a 5% residue level FDR over different search database sizes (error bars represent the 0.95 CI). (B) Total number of acquired MS2 spectra (averaged over three replica, dots show individual values). The stacked bars in the BSC bar indicate the mean number of spectra that were acquired with HCD (yellow, 95.2%) or EThcD (green, 4.8%). (C) Sequence coverage comparison of the PSMs corresponding to the cross-linked residue pairs from (A). (D) Performance comparison of the best method combinations for up to three injections of the same sample. Mean numbers of identified cross-linked residue pairs passing a 5% residue level FDR (error bars represent the 0.95 CI). Database size is 512 sequences.

References

    1. Paramelle D.; Miralles G.; Subra G.; Martinez J. Proteomics 2013, 13, 438–456. 10.1002/pmic.201200305. -DOI -PubMed
    1. Rappsilber J. J. Struct. Biol. 2011, 173, 530–540. 10.1016/j.jsb.2010.10.014. -DOI -PMC -PubMed
    1. Sinz A. Mass Spectrom. Rev. 2006, 25, 663–682. 10.1002/mas.20082. -DOI -PubMed
    1. Walzthoeni T.; Leitner A.; Stengel F.; Aebersold R. Curr. Opin. Struct. Biol. 2013, 23, 252–260. 10.1016/j.sbi.2013.02.008. -DOI -PubMed
    1. Aebersold R.; Mann M. Nature 2003, 422, 198–207. 10.1038/nature01511. -DOI -PubMed

Publication types

Grants and funding

LinkOut - more resources