Progress toward Proteome-Wide Photo-Cross-Linking to Enable Residue-Level Visualization of Protein Structures and Networks In Vivo

Abstract

Cross-linking mass spectrometry (XL-MS) is emerging as a 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 cross-linkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MScleavable cross-links), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-cross-linkers 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 are yet to be employed for proteome-wide studies because their products are challenging to identify. Here, we demonstrate the synthesis and application of two heterobifunctional photo-cross-linkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil doubly fissile MS-cleavable linkages upon acyl transfer to protein targets. Moreover, these cross-linkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-cross-linking in cellulo. These studies elucidate a small portion of Escherichia coli's interaction network, albeit with residue-level resolution. With further optimization, these methods will enable the detection of protein quinary interaction networks in their native environment at residue-level resolution, and we expect that they will prove useful toward the effort to explore the molecular sociology of the cell.

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).…”

“…Four leading structural proteomic methods include hydrogen–deuterium exchange (HDX), − methionine oxidation methods (e.g., SPROX), , fast photochemical oxidation of proteins (FPOP), , and limited proteolysis mass spectrometry (LiP-MS). − In these methods, regions within proteins that are solvent-accessible are labeled, respectively, by deuteration at backbone amides, by oxidation at methionine from H 2 O 2 , by oxidation at any amino acid from HO radicals, or by cleavage from a broad-spectrum protease . 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), − though it differs from the other methods in that it requires sequencing low-abundance cross-linked peptides, creating a unique set of technical challenges. , …”

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

“…11 Crosslinking 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][13][14][15] though it differs from the other methods in that it requires sequencing low-abundance crosslinked peptides from vast search spaces, creating a unique set of technical challenge. 16,17 Amongst 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, significant amplification of the search space). In its modern form (devised by Feng et al), the experimentalist subjects a complex mixture of proteins to a pulse of proteolysis with a non-specific protease (typically proteinase K) under native conditions, causing solvent-accessible or unstructured portions within proteins to get cleaved (Figure 1A).…”