Proteome-Wide Photo-Crosslinking Enables Residue-Level Visualization of Protein Interaction Networks in vivo

Abstract

Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo- XL-MS has a propensity to capture protein-protein interactions, particularly involving low- abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the "darker" corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.

Cross-linking mass spectrometry for mapping protein complex topologies in situ

“…However, due to the dilution in signal (due to the larger number of possible cross-links that can be formed) and a further increase in search space, these cross-linked peptides are often more difficult to identify. Only recently have some proof-of-concept studies shown that it is possible to use these cross-linkers in complex samples [ 29 ]. Translationally incorporated diazirine-containing photo-activatable amino acids are also available [ 30 , 31 ].…”

Silyl Ether Enables High Coverage Chemoproteomic Interaction Site Mapping

“…Numerous innovative methods including thermal protein profiling (TPP) 1 , limited proteolysis (LiP) [2][3][4] , stability of proteins from rates of oxidation (SPROX) 5 , covalent protein painting (CPP) 6 , and photoaffinity labeling (PAL) [7][8][9][10][11] have yielded proteome-wide portraits of protein-protein [12][13][14] , proteinoligonucleotide 15 , protein-lipid [16][17][18][19][20][21][22] , protein-metabolite 23 , protein-natural products [24][25][26][27] , proteinglycan 28 , protein-cofactor [16][17][18] , protein-small molecule 10,11,29 , and even protein-drug interactions [30][31][32][33][34] .…”

FLiPPR: A Processor for Limited Proteolysis (LiP) Mass Spectrometry Data Sets Built on FragPipe

“…11 Cross-linking mass spectrometry (XL-MS) is another method that can provide rich structural information in the form of residue−residue contacts (which can serve as distance restraints), 12−15 though it differs from the other methods in that it requires sequencing low-abundance crosslinked peptides, creating a unique set of technical challenges. 16,17 Among the labeling-based methods, LiP-MS has emerged as a popular structural approach because it has a few key advantages (e.g., residue-level resolution, proteome-wide coverage) without some drawbacks that affect other methods (e.g., need for specialized purpose-built instruments, need to identify rare low-abundance species). In its modern form (devised by Feng et al 9 ), the experimentalist subjects a complex mixture of proteins to a pulse of proteolysis with a broad-spectrum protease (typically Proteinase K, also referred to as PK) under native conditions, causing solvent-accessible or unstructured portions within proteins to get cleaved (Figure 1A).…”