Yi He (San Jose, CA / US), Erum Raja (Rockford, IL / US), Leigh Foster (Rockford, IL / US), Anthony Ciancone (Frederick, MD / US), Anneliese Faustino (Baltimore, MD / US), Piyoosh Sharma (Baltimore, MD / US), Stephen Fried (Baltimore, MD / US), Francis O’Reilly (Frederick, MD / US), Ryan Bomgarden (Rockford, IL / US), Rosa Viner (San Jose, CA / US)
Introduction
Cross-linking mass spectrometry (XL-MS) has grown dramatically as a key workflow for elucidating protein higher-order structure and mapping protein-protein or protein-nucleic acid interaction networks on a proteome-wide scale. Photoactivatable crosslinkers SDA and DizSEC (cleavable) link lysine with any residue in proteins, providing comprehensive information about protein structures. However, challenges remain in both instrument methods and data analysis for these crosslinkers. In this work, we optimized XL-MS workflows for SDA and DizSEC crosslinked samples and developed a new MS2-MS3 method to improve DizSEC XL identifications.
Methods
Heterobifunctional photoactivatable -crosslinkers SDA (succinimidyl 4,4'-azipentanoate), sulfo-SDA (sulfosuccinimidyl 4,4'-azipentanoate) and DizSEC (2,5-Dioxopyrrolidin-1-yl (2-(3-methyl-3H-diazirin-3-yl)ethyl)carbamate) were used to crosslink standard proteins. Separation was achieved using Thermo Scientific™ Vanquish™ Neo LC system with a 60 min gradient using EASY-Spray™ PepMap™ Neo column. Following separation, the peptides were detected on Thermo Scientific™ Orbitrap Ascend™ or Exploris 480 ™ mass spectrometers, and data were analyzed using XlinkX node in Proteome Discoverer v3.1 (Thermo Scientific) and xiSEARCH/xiFDR. Identified XLs were visualized using the XMAS plug-in for ChimeraX.
Results
Photoactivatable, heterobifunctional crosslinkers like SDA can link lysine residues with any residue providing detailed information for protein structures but also pose challenges due to lack of MS-cleavability and increased search space during data analysis. Recently, a new cleavable crosslinker, DizSEC, was developed to overcome this issue. DizSEC links the same residues as SDA, but DizSEC crosslinked peptides can generate unique peptide pairs in MS/MS to facilitate data analysis. We compared three photo-crosslinkers (SDA, sulfo-SDA, and DizSEC) using monomeric, dimeric, and tetrameric standard proteins. We developed three FAIMS- MS methods: MS2 SCE, MS2 SCE prioritizing charge states +4 to +6, and a new MS2-MS3 method for DizSEC. Compared to the standard MS2 SCE method, the instrument method prioritizing high charge states increased the number of crosslinks by 30-50%. When using the MS2-MS3 workflow for DizSEC, the instrument identifies peptide pairs with a 26 Da difference in MS/MS and selects them for MS3 fragmentation. The MS2-MS3 method generated similar numbers of crosslinks to the standard MS2 SCE method but improved FDR. For data analysis, we successfully created the new DizSEC templates in Proteome Discoverer 3.1 for both MS2 and MS2-MS3 data analysis. Three different SDA crosslinkers provided a similar number of crosslinked peptides for monomeric protein HSA; however, SDA and sulfo-SDA outperformed DizSEC for multimeric proteins probably due to the length of the linker.
Novel aspect
Optimized XL-MS workflows for SDA and the new cleavable photo-crosslinker DizSEC