Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure

Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, current in vivo XL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions for in situ XL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, high-concentration formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.


Introduction
Maps of protein-protein interactions (PPIs) generate networks that help us understand how cells function and respond to various stimuli.For 20 years or more, these networks have been sampled using affinity pulldown mass spectrometry (AP-MS) experiments [1][2][3] .However, AP-MS only generates indirect interaction data, and the experiments are prone to false positives.Crosslinking mass spectrometry (XL-MS) could produce a higher quality of interaction data by providing a distance measurement between two points in cellular space 4 .It is used very successfully for protein structure determination of reconstituted multiprotein complexes [5][6][7] , thus it should be useful for interactome mapping.Ideally, crosslinks would sample the undisturbed spatial proteome.In situ XL-MS experiments have already supported some compelling modeling and interaction mapping studies [8][9][10] , suggesting that in-depth PPI mapping may soon be possible.
Unfortunately the spatial proteome is not sampled very deeply by conventional XL-MS methods, although some progress is being made [11][12][13] .After crosslink installation, cells are lysed and digested in a standard "bottom-up" workflow common to conventional proteomics routines, generating linked peptides for detection by MS.Crosslinked peptides are minor reaction products and cannot be identified with the conventional database searching strategies used in the proteomics community.New software makes the detection of crosslinks more efficient than ever before [14][15][16][17][18] , but very little progress has been made in improving the crosslinking reaction itself.
To faithfully sample both the spatial and temporal properties of the proteome, crosslinkers must cross the cell membrane, perfuse freely throughout the cell, and react quickly.The crosslinkers most readily available to the community target lysines through N-hydroxysuccinimide (NHS) esters, but the majority of these reagents do not permeate the membrane very well at all 19 .As a result, incubation times as long as an hour are needed to integrate a detectable level of reaction products.The structural proteome could very well be distorted during this timeframe, as cells are dynamic and respond to chemical stimuli.These issues may be addressed by increasing the biocompatibility of the reagents 19,20 , but the design constraints are high: maximum crosslink yields must be realized in as short a time as possible.
New faster-acting reagents are not the only way to approach the problem.It would be ideal to stabilize the spatial proteome first: arresting all protein movement and uncoupling cellular dynamics from crosslinker reaction dynamics.Stabilization would permit longer crosslinker reaction times and provide more control over crosslinking yield.If done quickly, it could even capture flux in the interactome.Formaldehyde-based fixation is a compelling stabilization option.Formaldehyde has been used for over 100 years to fix cells for microscopy and preserve biological samples for long-term tissue storage 21,22 .It too is a crosslinker, but it is quite different from the reagents used in XL-MS.Formaldehyde is very water soluble, is rapidly taken up into cells, and its reaction kinetics are fast 23,24 .The mechanism of stabilization is complex.At a common fixative concentration of 4% (1.3M), formaldehyde exists in equilibrium between reactive free formaldehyde and unreactive methylene glycol, the latter in large excess 25,26 .Fixation appears to involve an initial burst phase (seconds to minutes) where formaldehyde crosslinks different classes of biological macromolecules, including proteins 23 .
Long-term stabilization is thought to involve the slow conversion of methylene glycol to reactive formaldehyde in a type of "clock" reaction 27 .The burst phase provides the stabilization needed for methods like immunofluorescence that are used to explore cellular mechanisms, whereas the extended incubations support tissue preservation for biobanks.Early tests of formaldehyde as a reagent for XL-MS tried to leverage this initial burst phase but were generally unsuccessful 28,29 .
A recent study uncovered a double methylene bridge between lysines that could exploited, but the yields from such reactions appear much lower than NHS-based crosslinkers 30 .In addition, the apparent nonselective nature of formaldehyde crosslinking would complicate an already challenging database search space 31 .
While it is not yet a viable crosslinker for in vivo XL-MS, it is clearly an effective stabilizer of protein and cellular structures.Very minimal formaldehyde crosslinking is already used to slightly stabilize protein complexes for many applications, including crosslinking 32,33 .
High concentrations are avoided over concerns with reagent competition.but because formaldehyde crosslinks are hard to detect, we reasoned that fixation even at high concentration may not strongly interfere with lysine-directed crosslinking reagents.This may seem counterintuitive based on our current understanding of formaldehyde chemistry.However, the initial stabilization of cells must only involve a very small fraction of amines directly associated with interfaces, thus leaving ample room for post-fixation crosslinking.Here, we describe an in situ XL-MS method that uses conventional formaldehyde fixation protocols to stabilize cells prior to the introduction of NHS-based crosslinkers.Surprisingly, fixation does not interfere with secondary crosslinking reactions, and more importantly, it allows us to develop methods that boost crosslinking yields for in situ work.

Results and Discussion
Preserving the spatial proteome for XL-MS.Slow crosslinking reactions can distort the equilibrium state of proteins and prevent accurate modeling of protein structures 34 ; thus we first examined if these slow reactions would also distort the spatial proteome.We chose to monitor the dynamic actin cytoskeleton in A549 cells, a human epithelial non-small cell lung cancer cell line.The actin cytoskeleton is an essential component of many cellular processes and thus a good indicator of spatial integrity 35 .We adopted a standard protocol for visualizing cells, involving fast fixation with 4% formaldehyde and staining with CF647-phalloidin, which labels filamentous actin (F-actin) by binding at the interface between F-actin subunits (Supplementary Figure 1A).Images show the striated patterns and absence of puncta that are expected for a healthy and stable cell (Fig. 1A,B).These images demonstrate the speed of fixation achievable with formaldehyde.
We then did an experiment where DSS, a widely used cell-permeable crosslinker, was applied at a typical concentration of 1 mM before fixation and image analysis.Significant distortions of the proteome were observed, visibly affecting ~70% of cells.Actin filaments were depolymerized and a large number of puncta were formed (Fig. 1A,B).Given the limited solubility of the reagent, the crosslinker is normally prepared as a dilution from a DMSO stock.Thus, we next tested the effect of the vehicle alone (2% DMSO) and observed an even higher level of distortion (Fig. 1A,B).Brightfield images of live cells treated with the vehicle revealed apoptosis in many of the cells (Supplementary Figure 1B).There was very little cellular distortion when we reversed the process: first fixing the cells with 4% formaldehyde, washing them to remove excess formaldehyde, and then crosslinking with DSS (Fig. 1A,B).Thus, conventional methods for in situ XL-MS appear to strongly distort the spatial proteome.Organic solvents are often needed for crosslink reagent solubilization and while the crosslinkers themselves can reduce cellular distortion to a degree, pre-fixing with a much faster-acting reagent like formaldehyde allows us to separate the stabilization phase from the crosslinking phase.Labelling the stabilized spatial proteome.However, we cannot assume that DSS even labels protein after prefixation.To explore this, we used N-(propionyloxy)succinimide, a monovalent NHS ester, as a surrogate for DSS.Single labeling events are much easier to detect and quantitate than crosslinking events, allowing us to measure a percent labeling of the entire proteome (see methods).Surprisingly, the application of formaldehyde had no effect on the level of reagent incorporation, even up to 4%, and results are independent of cell type (Fig. 2A and Supplementary Figure 2A,B).Labeling was extensive.Labels were incorporated across the detectable dynamic range of the proteome and exhibited no major bias (Fig. 2C,D and Supplementary Figure 2C).These results are entirely dependent on the washing step, however.
Lysine labeling was completely suppressed in the presence of formaldehyde (Supplementary Figure 2D), consistent with the formation of a methylol derivative and/or a Schiff base 23 , which can be reversed by washing the cell before applying DSS.These results indicate that the pool of free amines is largely unchanged by formaldehyde-based fixation, suggesting that the crosslinking experiment on the fixed cells was successful (Fig. 1).
Before confirming this conclusion, we explored how fixation could improve the permeability of crosslinking reagents, again using monovalent NHS esters as surrogates.It is standard practice in immunofluorescence to use surfactants like Triton-X 100 to permeabilize cells.These surfactants insert into the lipid bilayer and partially erode the integrity of the membrane.Common surfactant concentrations for washing-in fluorescent stains and antibody conjugates are 0.1-0.5%.Here, we fixed A549 cells with 4% formaldehyde and then permeabilized with 0.1% Triton-X 100, on the low end of the scale.We chose biotin-X-NHS as a surrogate for a larger crosslinker and sulfo-NHS-LC-biotin as a surrogate for a charged crosslinker, which should not permeate the membrane at all.There was no detectable labeling of the proteome in the non-permeabilized cells for either reagent, as expected.The labeling levels increased substantially upon fixation, presumably because formaldehyde at this concentration has a mild permeabilization effect 36 .The labeling increased even further with surfactant-based permeabilization (Fig. 2B).Fixation with permeabilization supported longer reaction times, even multiple sequential reagent additions to build up labelling levels, all with no major distortion of the spatial proteome (Supplementary Figure 3).Taken together, these findings suggest that the full range of chemical crosslinkers could be applied to a pre-stabilized spatial proteome.Crosslinking of fixed cells.We then explored how pre-fixation influences the crosslinking reaction itself, using the workflow shown in Fig. 3.This strategy was designed to compare fixed and unfixed reaction conditions and provide a sufficient depth of coverage to determine the influence of pre-fixation on relative yields.We again chose DSS for the comparison.It is a common crosslinker, but one that generates only modest in situ crosslinking yields in conventional XL-MS experiments 37 .Our results confirmed this (Fig. 4A).In a 30 min reaction, less than 30,000 crosslink spectrum matches (CSMs) were detected from live A549 cells, corresponding to 1,348 unique crosslinks at a 5% FDR.Most of these crosslinks are intra-protein linkages as expected and we observed ratios of reaction products similar to those generated for free proteins and complexes 38 .(See Table S1 for a complete breakdown of reaction products for 5% and 1% FDR.) Figure 3. Workflow for formaldehyde pre-stabilization followed by in situ XL-MS.Cells are fixed with 4% formaldehyde for 10 minutes.After fixation, excess formaldehyde is washed away prior to the introduction of the crosslinker.After secondary crosslinking, cells are collected, lysed, and formaldehyde linkages are reversed by boiling.Extracted protein is then cleaned up via an SP3 44 protocol and digested overnight with trypsin.Peptides then undergo high-pH fractionation for LC-MS/MS data acquisition.Data were then processed using pLink 2 17 .Created with Biorender.com.
Fixation alone was insufficient to enhance yields for DSS, even though it increased the labeling yield of monomeric labeling agents.Without permeabilization, we only observed a 1.2fold increase in CSMs and no change in the number of unique crosslinks (Fig. 4A).However, after treating fixed cells with 0.1% Triton-X 100, the number of CSMs and unique crosslinked peptides increased almost two-fold (Fig. 4A).The overall quality of these identifications is superior to the standard analysis in both yield and score, and similar ratios of reaction products are observed (Supplementary Figure 4 and Table S1).These conditions generated a modest 141 PPIs, (Fig. 4B), so we attempted to increase yield through multiple applications of DSS.We chose three sequential 1 mM treatments with DSS based on our observation that a 3X application retained ultrastructure (Supplementary Figure 3C).Multiple treatments resulted in a three-fold increase in CSMs and a four-fold increase in unique crosslinks, compared to live cells (Fig. 4A).
The yield of PPIs also increased over 3-fold to 448 (Fig. 4B and Table S1).We observed that multiple treatments also improved the quality of the detected PPIs: STRING scores were 0.9 for most hits (Fig. 4C).At this level of interactome sampling, which is biased towards more abundant proteins given our sample fractionation strategy, most of the PPIs that we detected should be represented in existing databases.Taken together, fixation with permeabilization enables a flexible crosslinking protocol where yields can be controlled and amplified where needed.Crosslinking of fixed cells -PhoX enriched.Although we can increase the yield of crosslinking reactions significantly over conventional methods, detection still benefits from selective enrichment of the reaction products.One of the most effective strategies involves isolation through an affinity tag in the spacer group situated between the two reactive centers, but there are few such reagents that efficiently cross the membrane.Fixation followed by permeabilization should allow any crosslinker to be used for in situ XL-MS, with perhaps only slight adjustment of the surfactant concentration.To explore this idea, we tested the PhoX crosslinker in a 1X treatment.This reagent contains a negatively-charged phosphonate that can be enriched by immobilized metal ion affinity chromatography (IMAC) resins 39 , in a very simple and robust process that is used for several applications in proteomics.However, the negatively charged phosphonate renders the molecule membrane-impermeable, restricting the crosslinker to lysates mostly, although derivatized versions of PhoX have been developed recently with improved penetrance 20 .
Fixation with permeabilization enabled an effective in situ reaction.We recovered approximately 13% (wt percent) of the total peptide digest in this experiment, of which 53% were crosslinked peptides of all types, 8% were verified phosphopeptides, and 39% were putative phosphopeptides and/or free peptides.These enrichment statistics are consistent with a previous crosslinking study 39 .We detected 168,177 CSMs in this data set, which reduced to 10,722 unique crosslinked peptides at a 5% FDR (Fig. 5A and Table S1).Yields are approximately two times greater than the 3X DSS crosslinking experiment in both categories (Fig. 4A), reducing to 499 unique PPIs at a 5% FDR.As with the 3X DSS reaction, most of the detected PPIs were matched to the STRING database with high scores (Fig. 5B, Table S2).A more in-depth analysis of the interprotein crosslinks showed a set of complexes that are consistent with the MS sampling depth that we applied (~5 microgram over 12 fractions) (Fig. S3).That is, most protein interactions that we detect involve relatively high abundance proteins, including subunits of the TRiC/CCT molecular chaperone, the 26S proteasome particle, the ribosome, histones, and the DNA replisome (Fig. 5C,D).However, additional complexes comprised of proteins with lower copy numbers are also in evidence, including the Ku70/80 heterodimer (Fig. 5E) involved in DNA damage repair, histone regulatory complexes such as histone deacetylase 2 interacting with REST corepressor 1, and the serine/threonine kinase Nek1 interacting with histone 2A.The latter is most interesting as it highlights that fast fixation with secondary crosslinking can capture transient enzyme-substrate complexes.Mapping the unique crosslinks to known protein complexes generated a histogram of distances that reflect good sampling of structure: 52% of crosslinks were within 20Å and 73% of crosslinks within 35Å (Fig. 5F) with few overlength crosslinks observed.

5C, Table
The depth of sampling was sufficient to begin exploring interactors that possess little available structural information.For example, we detected an interaction between apoptosis inhibitor 5 (API5) and the apoptotic chromatin condensation inducer in the nucleus (ACIN1), a nuclear complex that regulates apoptotic DNA fragmentation 40 .In our data, the complex was mapped with 2 interprotein crosslinks and 15 intraprotein crosslinks.We generated structures of the dimer using AlphaFold2 multimer 41,42 and found that 52% of crosslinks were within 20Å and 76% of crosslinks within 35Å (Supplementary Figure 5).In all models, interlinks were mapped at distances below 20Å, whereas several of the overlength intraprotein crosslinks span nominally disordered regions.This example highlights the potential benefit of in situ crosslinking as these linkages could be used to drive a more authentic modeling effort.
In summary, standard in vivo crosslinking protocols can distort the spatial proteome and should be used with caution, although the degree to which spurious PPIs are generated is currently unknown.We show that pre-fixing cells is effective at stabilizing the proteome for in situ XL-MS experiments.Together with permeabilization routines, it supports extended reaction times, higher crosslinking yields, and widens the scope of crosslinkers that can be used.It is somewhat surprising that a formaldehyde-based pre-stabilization method would support XL-MS at all, especially at the elevated concentrations we used (4%).Formaldehyde fixation involves the irreversible crosslinking of free amines, at least based on many classical bioconjugation texts 43 .However, we demonstrate that most of the modifications arising from a typical fixation experiment are reversible, simply by washing the treated cells with buffer.More recent literature has demonstrated that even terminal lysine-specific reaction products have a range of stabilities.
For example, hydroxymethylated and bridged amines are the major products from short-term fixation, which have been shown to be reversible in NMR studies of free amino acids, whereas N-methylation and N-formylation are not 31 .How then does fixation occur?It would seem to be the product of a broad reaction profile involving multiple different protein residues, and perhaps other biomolecules 23 .But fixation clearly does not require extensive lysine-mediated crosslinking, otherwise labeling yields would be negatively affected.We did not observe such a drop, except under extended fixation times (where N-methylation and N-formylation were indeed observed).
Rapid pre-fixation of cells restores flexibility to method development for in situ XL-MS.
Reagents can now be designed for effective PPI mapping without concern over membrane permeability and reaction times.This development will help in situ XL-MS become a viable alternative to AP-MS techniques for interactome analysis.Because it is based upon an established microscopy technique for mapping the spatial distribution of proteins, this method should also promote confidence in associations detected by in situ XL-MS.onto microscope slides with EverBrite + DAPI mounting medium (Biotium) and sealed with clear nail polish.Cells were imaged with an AxioObserver inverted microscope using the 40X oil immersion objective and imaged in the 647nm and DAPI channels using the ZEN microscopy software.Fluorescent phalloidin micrographs were then evaluated for disruptions to the spatial proteome post-treatment.The features we tracked to investigate structural perturbations were straited actin filaments, general actin structures, the presence of filopodia in confluent regions, and rounded cells.Approximately 100 cells were inspected across 6-7 micrographs, for each treatment.Images pseudo-coloured, with brightness adjusted for merged composites using ImageJ software.
Peptides were added to beads at a 1:10 (μL beads:μg peptide) and incubated at ambient temperature for 30 minutes under gentle rotation.Beads were then washed three times with 80% MeCN + 0.1% TFA at 4X bead volume, and once with H2O at 4X bead volume.Peptides were eluted from beads twice with 5% ammonium hydroxide at 2X bead volume for two minutes each time.

High-pH fractionation of crosslinked peptides
Crosslinked peptides were resuspended in mobile phase A (20mM ammonium formate, pH 10) and were loaded onto an Agilent 1260 infinity II system.Peptides were accumulated onto a ZORBAX RRHD Extended-C18 column (80Å pore size, 2.1 x 150mm, 1.8μm particles, Agilent) at 50°C.Samples were eluted at a flow rate of 0.2mL/min using a 54 minute multistep gradient from 5% mobile phase B (100% MeCN) for 6 minutes, then 5-45% B for 34 minutes followed by a hold at 45% B for 5 minutes, then a ramp of 45-80% B for 1 minute with a hold at 80% B for 4 minutes and finally a ramp of 80-5% B for 4 minutes.Fractions were collected every 1.2 minutes and concatenated from 48 to 12 fractions following a concatenation scheme of fractions 1+13+25+37, fractions 2+14+26+38,…, and so on.

LC-MS/MS analysis of monovalent labeled peptides
Monovalent-labeled peptides were resuspended in mobile phase A (0.1% formic acid) and loaded onto a Vanquish Neo HPLC coupled to a nano-ESI source of an Orbitrap Eclipse (ThermoFisher Scientific).Samples were injected onto a 300μm x 5mm PepMap Neo Trap Cartridge peptide trap column (C18, 5μm particle size, 100Å pore size, ThermoFisher Scientific) and separated on an EASY-Spray 75μm x 50cm PepMap HPLC column (C18, 2μm particle size, 100Å pore size, ThermoFisher Scientific) at a flow rate of 300 nL/min at 40°C using a multistep gradient from 2-30% mobile phase B (80% MeCN in 0.1% formic acid) for 75 minutes, 30-45% B for 45 minutes, 45-99% B for 1 minute, and a 10 minute wash at 99% B. A typical datadependent analysis used a full MS scan of m/z 375-1800, selecting charge states of 2-6+ for fragmentation and scanning in the orbitrap.MS 1 and MS 2 scan resolutions were 120,000 and 30,000, respectively.For MS 2 , samples were isolated with a m/z 1.2 window and underwent a normalized collision energy for stepped-HCD fragmentation of 27, 30, and 33%.Maximum injection time was set to 50 and 54 milliseconds for MS 1 and MS 2 , respectively, and dynamic exclusion was set for 30 seconds.

LC-MS/MS analysis of crosslinked peptides
Fractionated crosslinked peptides were loaded onto a Vanquish Neo HPLC coupled to a nano-ESI source of an Orbitrap Eclipse (ThermoFisher Scientific).Samples were injected onto a 300μm x 5mm PepMap Neo Trap Cartridge peptide trap column (C18, 5μm particle size, 100Å pore size, ThermoFisher Scientific) and separated on an EASY-Spray 75μm x 50cm PepMap HPLC column (C18, 2μm particle size, 100Å pore size, ThermoFisher Scientific) at a flow rate

Crosslink data analysis
Crosslink data were analyzed on pLink 2.3.11 17with the following parameters: minimum peptide length set to 6; maximum peptide length set to 60; maximum of 3 missed cleavages for trypsin; precursor mass tolerance set as 5 ppm; fragment mass tolerance set as 10 ppm; carbamidomethylation of cysteine (57.021 u) set as fixed modification; oxidation of methionine (15.995 u) set as variable modification.Crosslink masses for DSS and PhoX on lysine were set as 138.068 u and 209.971 u, respectively; monolink masses for DSS and PhoX on lysine were set as 156.079 u and 277.982 u, respectively.Data were searched against the full human proteome (retrieved from Uniprot on February 13, 2023).Results are reported at a 1% and 5% FDR set at the PSM level, with FDR calculations for intra-protein and inter-protein crosslinks evaluated separately.

Crosslink mapping to protein complexes and PPI network analysis
PhoX crosslinks were mapped using xiVIEW 45 onto select human protein complexes.Predicted models of the structure for the API5-ACIN1 complex was generated using AlphaFold2multimer 42 on COSMIC2 41 .Crosslinks with distances were exported as a "PyMOL command file" which was then imported into PyMol version 2.5.8 for visualization.PPIs generated from pLink crosslink spectra output for the first considered interaction and were visualized on Cytoscape version 3.10.1.The human PPI database was downloaded from STRING (https://string-db.org/) 47for analysis of PPI confidence using an in-house python script.

Data Availability
All raw data are available via the PRIDE partner repository 48 with the dataset identifier PXD051075  Figure 3. Workflow for formaldehyde pre-stabilization followed by in situ XL-MS.Cells are fixed with 4% formaldehyde for 10 minutes.After fixation, excess formaldehyde is washed away prior to the introduction of the crosslinker.After secondary crosslinking, cells are collected, lysed, and formaldehyde linkages are reversed by boiling.Extracted protein is then cleaned up via an SP3 44 protocol and digested overnight with trypsin.Peptides then undergo high-pH fractionation for LC-MS/MS data acquisition.Data was then processed using pLink 2 17 .Created with Biorender.com.

Figure 1 .
Figure 1.Conventional in situ XL-MS distorts cell structure.(A) Fluorescent imaging of actin cytoskeleton (Green) and DNA (blue) in A549 cells for formaldehyde-preserved cells, live cells treated with 1mM DSS (2%DMSO), live cells treated with 2% DMSO, and formaldehyde-preserved cells treated with 1 mM DSS (2% DMSO).(B) Cells counted for presence (orange) or absence (blue) of cellular disruptions during respective treatments (n ≥ 100 cells per treatment).See methods for indicators of disruption.

Figure 2 .
Figure 2. Pre-stabilizing cells with formaldehyde does not impact protein labeling.(A) Average percent proteome labeling using N-(propionyloxy)succinimide across E. coli and human cells with increasing concentrations of formaldehyde.(B) Comparison of percent proteome labeling with biotin-X-NHS (blue) and sulfo-NHS-LC-biotin (orange) in A549 cells,.forlive, fixed, and fixed + permeabilized states.(C) Histogram of identified protein

Figure 4 .
Figure 4. Effect of fixation and permeabilization on in situ XL-MS.(A) Number of CSMs (blue) and unique crosslinks (orange: loop-links, grey: intraprotein, and yellow: interprotein) identified from in situ crosslinking with DSS, in either live cells, fixed cells, fixed and permeabilized cells, or fixed cells with a 3X DSS treatment.(B) Number of PPIs identified from in situ crosslinking with DSS, in either live cells, fixed cells, fixed and permeabilized cells, and fixed cells with a 3X DSS treatment.(C) STRING score distribution for STRING PPIs (grey), live cells (red), fixed cells (orange), fixed + permeabilized cells (yellow), and fixed cells with a 3X DSS treatment (blue); PPIs not found in STRING database labeled as not-found (N/F).

Figure 5 .
Figure 5.In situ crosslinking of fixed and permeabilized A549 cells with PhoX.(A) Number of CSMs (blue) and unique crosslinks (orange: loop-links, grey: intraprotein, and yellow: interprotein) identified from in situ crosslinking with PhoX, in fixed and permeabilized cells.(B) STRING score distribution for STRING PPIs (blue) and in situ PhoX crosslinked cells (orange); PPIs not present in STRING database labeled as not-found (N/F).(C) PPI network plot of all detected interactions from in situ PhoX crosslinking.(D) Crosslinks mapped to the McM -DNA replisome (mapped to PDB 7PLO) and (E) Ku70/Ku80 (mapped to PDB 1JEQ).(F) Histogram of Cα-Cα distances of PhoX crosslinks mapped to all known structures.

Figure 1 .
Figure 1.Conventional in situ XL-MS distorts cell structure.(A) Fluorescent imaging of actin cytoskeleton (Green) and DNA (blue) in A549 cells for formaldehyde-preserved cells, live cells treated with 1mM DSS (2%DMSO), live cells treated with 2% DMSO, and formaldehydepreserved cells treated with 1 mM DSS (2% DMSO).(B) Cells counted for presence (orange)/absence (blue) of cellular disruptions during respective treatments (n ≥ 100 cells per treatment).See methods for indicators of disruption.

Figure 2 .
Figure 2. Pre-stabilizing cells with formaldehyde does not impact protein labeling.(A) Average percent proteome labeling using N-(propionyloxy)succinimide across E. coli and human cells with increasing concentrations of formaldehyde.(B) Comparison of percent proteome labeling with biotin-X-NHS (blue) and sulfo-NHS-LC-biotin (orange) in A549 cells,.forlive, fixed, and fixed + permeabilized states.(C) Histogram of identified protein abundance from human non-labeled lysate (light-orange), fixed only (purple), and (D) fixed + permeabilized (red), labeled with biotin-X-NHS.Protein abundancies retrieved from PaxDb 46 .

Figure 4 .
Figure 4. Effect of fixation and permeabilization on in situ XL-MS.(A) Number of CSMs (blue) and unique crosslinks (orange: loop-links, grey: intraprotein, and yellow: interprotein) identified from in situ crosslinking with DSS, in either live cells, fixed cells, fixed and permeabilized cells, or fixed cells with a 3X DSS treatment.(B) Number of PPIs identified from in situ crosslinking with DSS, in either live cells, fixed cells, fixed and permeabilized cells, and fixed cells with a 3X DSS treatment.(C) STRING score distribution for STRING PPIs (grey), live cells (red), fixed cells (orange), fixed + permeabilized cells (yellow), and fixed cells with a 3X DSS treatment (blue); PPIs not found in STRING database labeled as not-found (N/F).

Figure 5 .
Figure 5.In situ crosslinking of fixed and permeabilized A549 cells with PhoX.(A).Number of CSMs (blue) and unique crosslinks (orange: loop-links, grey: intraprotein, and yellow: interprotein) identified from in situ crosslinking with PhoX, in fixed and permeabilized cells.(B) STRING score distribution for STRING PPIs (blue) and in situ PhoX crosslinked cells (orange); PPIs not present in STRING database labeled as not-found (N/F).(C) PPI network plot of all detected interactions from in situ PhoX crosslinking.(D) Crosslinks mapped to McM -DNA replisome (mapped to PDB 7PLO) and (E) Ku70/Ku80 (mapped to PDB 1JEQ).(F) Histogram of Cα-Cα distances of PhoX crosslinks mapped to structures