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xIP-MS: topological analysis of chromatin-associated protein complexes using single affinity purification



In recent years, cross-linking mass spectrometry (XL-MS) has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, XL-MS workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase (PRPP) complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures.
xIP-MS: streamlined structural proteomics
xIP-MS: topological analysis of chromatin-associated protein complexes using single
affinity purification
Matthew M Makowski¹, Esther Willems¹, Pascal WTC Jansen¹, Michiel Vermeulen¹˒²˒*
1. Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert
Grooteplein 28, 6525 GA Nijmegen, the Netherlands
2. Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG Utrecht, the
Running Title: xIP-MS: streamlined structural proteomics
Keywords: Chemical cross-linking mass-spectrometry, chromatin, affinity purification
*Correspondence addressed to M.V. at:
MCP Papers in Press. Published on November 11, 2015 as Manuscript M115.053082
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
xIP-MS: streamlined structural proteomics
XL-MS Cross-linking Mass Spectrometry
xIP-MS Cross-linking Immunoprecipitation Mass Spectrometry
coIP Co-immunoprecipitation
PRPP Phosphoribosyl Pyrophosphate Synthetase Complex
SMC Structural Maintenance of Chromosomes
XRCC X-Ray Repair Cross-Complementing Protein
MCM Mini-Chromosome Maintenance
LFQ Label free quantification
DSB Double-strand breaks
PTM Post-translational modification
GFP Green fluorescent protein
SCX Strong cation exchange
SEC Size exclusion chromatography
xIP-MS: streamlined structural proteomics
In recent years, cross-linking mass spectrometry (XL-MS) has proven to be a robust and
effective method of interrogating macromolecular protein complex topologies at peptide
resolution. Traditionally, XL-MS workflows have utilized homogenous complexes obtained
through time-limiting reconstitution, tandem affinity purification, and conventional
chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS),
a simple, rapid, and efficient method for structurally probing chromatin-associated protein
complexes using small volumes of mammalian whole cell lysates, single affinity purification,
and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS
using the structurally well-characterized phosphoribosyl pyrophosphate synthetase (PRPP)
complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6
(Ku70/86), and MCM complexes, and we provide novel structural and biological insights into
their architectures and molecular function. Of note, we use xIP-MS to perform topological
studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently
straightforward and efficient to allow comparative cross-linking experiments. This work,
therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for
interrogating chromatin-associated protein complex architectures.
xIP-MS: streamlined structural proteomics
The structural basis of specific protein-protein interactions and higher-order protein complex
multimerization crucially informs our understanding of many molecular and cellular processes.
AP-MS-based strategies have been a major player in the elucidation of specific protein-protein
interactions and core protein complexes [1-3]. However, while quantitative AP-MS/MS
experiments identify specific interactors for any given bait, they cannot differentiate between
direct and indirect interactors or provide structural or topological information on protein complex
assemblies. Furthermore, traditional structural methodologies often fail with large or dynamic
protein complexes including chromatin remodeling or chromatin associated protein complexes.
Cross-linking mass spectrometry (XL-MS), therefore, has emerged as a powerful technique for
analyzing protein complex architectures through direct observation of subcomplex interfaces at
the peptide level [4-6]. Landmark studies used XL-MS to reveal the architectures of the
chromatin remodeling complexes Ino80 and SWR1 [7, 8], and other chromatin-associated
complexes have been interrogated using traditional XL-MS workflows [9-14].
In general, cross-linked peptides represent a small fraction of the total pool of peptides measured
in an MS/MS analysis. Therefore, traditional XL-MS workflows have utilized homogenous
complexes obtained through time-limiting reconstitution, tandem affinity purification, and
conventional chromatography workflows to facilitate the detection of low abundance cross-
linked peptides from the target protein complex. For example, reconstitution of complexes to
near homogeneity using tandem tagging systems has been previously reported, using a variety of
tagging combinations [13, 15, 16]. Similarly, baculovirus over-expression and recombinant
yeast systems have also been used effectively to maximize protein yield [7-9, 14]. Further
strategies often used to enrich cross-link identifications include strong cation exchange
xIP-MS: streamlined structural proteomics
chromatography (SCX), size exclusion chromatography (SEC), the use of multiple proteases, or
alternate cross-linking strategies including acid-acid cross-linkers, CID-cleavable cross-linkers,
or tagged, enrichable cross-linkers [16-20]. These steps have been necessary to enrich to an
appreciable degree the number of measurable cross-linked peptides from the target complex.
However, implementing and optimizing these workflows represents a considerable barrier to
entry to the XL-MS field for many molecular biologists.
Single-AP-MS strategies are commonly applied in chromatin biology, where specific interactors
can be identified in the presence of a vast amount of background proteins [2, 3, 21-23]. Our
group and others have previously established workflows for identifying specific protein
interactors and interaction stoichiometries using a GFP-tagging BAC transgene systems
expressing tagged baits at a near-endogenous level [22, 24-26]. However, thus far simple, single-
IP methodologies have not been widely used in XL-MS experiments. One main challenge is
sufficiently purifying protein complexes to facilitate the consistent identification of low-
abundance cross-linked peptides over background. For example, a recent study used specially
engineered high-affinity, lysine-free GFP-nanobodies to efficiently extract endogenous level
tagged baits [27]. Here, we present cross-linking immunoprecipitation mass spectrometry, or
xIP-MS, utilizing a simple, efficient GFP-AP workflow followed by a high-stringency washing
procedure to obtain highly pure protein complex from small volumes of human whole cell
lysates. xIP-MS couples single-AP GFP purification with on-bead crosslinking and sample prep
for MS/MS analysis. Dynamic exclusion settings, exclusion of lower charge-state peptides, and
the high speed of modern mass spectrometers facilitate the fragmentation and identification of
low abundance cross-linked peptides. Furthermore, computational analysis of cross-linked
peptides is a crucial and widely discussed aspect of XL-MS workflows [28-30]. In xIP-MS, we
xIP-MS: streamlined structural proteomics
use straightforward data-filtering steps based on peptide length (selectively removing those
matches where one long, high scoring peptide compensates for a short, ambiguous peptide
match) and reproducibility in multiple experiments (to remove spurious matches) to increase
confidence in cross-link identifications.
We benchmark xIP-MS against the structurally well-characterized PRPP complex and provide
evidence for a structural ensemble of PRPP complexes in vivo [31]. We then apply xIP-MS to
the chromatin-associated cohesin, XRCC5/6, and MCM complexes. We use xIP-MS to analyze
the cohesion complex and observe numerous cross-links within the well-ordered head region,
complementing previous cross-linking experiments and directly supporting a model for the
human cohesin head interaction [11, 12]. Next, we used xIP-MS to analyze conformational
changes in the x-ray repair cross-complementing protein 5/6 complex (XRCC5/6, or Ku70/86)
[32]. We observe conformational flexibility in the Ku and SAP domains consistent with their
involvement in DNA binding during double-stranded break repair [33]. Finally, we use xIP-MS
to characterize soluble mini-chromosome maintenance (MCM) subcomplexes [34-37]. We
present an architecture for interactions between MCM subunits and show that soluble MCM
subcomplexes are generally cell-cycle invariant.
The xIP-MS workflow requires approximately the time and resources of a standard coIP
experiment, yet can provide additional structural information about subunit interfaces and
complex topologies. We further demonstrate the biological usefulness of xIP-MS in studying
complex structural ensembles, conformational flexibility, or subcomplex architectures under
perturbation. Thus, we present here xIP-MS as a simple, efficient, and broadly applicable
technique for probing protein complex architectures.
xIP-MS: streamlined structural proteomics
Cell culture and lysate collection
Cell lysates were collected from stably transgenic HeLa Kyoto cells expressing near-endogenous
levels of GFP-tagged bait protein from a recombineered BAC transgene system [24, 25]. Cell
lines were cultured in DMEM plus 10% FBS and 100U/ml penicillin and streptomycin. For one
week, cells were kept under selection with 400ug/ul geneticin to ensure stable integration of the
tagged bait, then expanded in normal media. Cell lysates were collected by resuspending cells in
five cell pellet volumes of lysis buffer (150mM NaCl, 50mM Tris pH 8.0, 1mM EDTA, 20%
glycerol) supplemented with 1% NP-40, 1mM DTT, and Roche EDTA-free complete protease
inhibitors (CPIs). Cell lysates were rotated at 4C for two hours. The lysate was then centrifuged
for 30 minutes at 4,000rcf and 4C, and the supernatant was collected and snap-frozen. This lysis
method produced high-quality lysates of ~10mg/mL protein concentration.
GFP affinity purification for label free quantification (LFQ)
20uL GFP bead slurry was used per affinity purification (Chromotek). Beads were pre-washed
three times with Buffer C (300mM NaCl, 20mM HEPES pH 7.9, 20% glycerol, 2mM MgCl2,
0.2mM EDTA) supplemented with 1% NP-40, 0.5mM DTT, and EDTA-free Complete Protease
Inhibitors (CPIs, Roche). 400ug whole cell lysate was added to the pre-washed beads and
adjusted to 400uL with whole cell lysis buffer plus 1% NP-40, 1M DTT, and CPIs. Ethidium
bromide was added to 50ug/mL. Reactions were incubated on a rotating wheel for one hour at
4C. After IP, beads were washed twice with Buffer C increased to 1M NaCl and supplemented
with 1% NP-40, 0.5mM DTT, and CPIs. Beads were then washed twice with PBS supplemented
with 1% NP-40, and finally washed twice with PBS. All supernatant was removed carefully with
xIP-MS: streamlined structural proteomics
a 30G syringe before sample prep for AP-MS/MS analysis. Control samples were prepared using
binding control agarose beads with the same protocol (Chromotek). All LFQ experiments were
performed in triplicate. GFP-AP and control samples from the same experiment were prepared
on the same day and analyzed by AP-MS/MS sequentially.
GFP affinity purification for xIP-MS
GFP affinity purifications for xIP-MS were performed essentially as described for LFQ GFP
affinity purifications. 30uL of GFP bead slurry was used per affinity purification, and 1mL of
total whole cell lysate was used per pulldown (~10mg/mL). All xIP-MS experiments were
performed in duplicate. Replicates were performed independently and measured by AP-MS/MS
On-bead chemical cross-linking for xIP-MS
On-bead cross-linking was performed by immediately resuspending beads in 50mM borate-
buffered saline containing 1mM BS3 (Thermo Scientific) following GFP affinity purification.
Cross-linking reactions were performed for one hour at room temperature with shaking at
1000rpms. Reactions were quenched by adding 100mM ammonium bicarbonate and incubated at
room temperature for ten minutes with shaking at 1000rpms. All supernatant was again carefully
removed with a 30G syringe.
Sample preparation for AP-MS/MS analysis
Sample preparation was performed in the same manner for LFQ and xIP-MS samples. Purified
or cross-linked proteins were denatured and reduced in elution buffer (2M urea, 100mM
xIP-MS: streamlined structural proteomics
ammonium bicarbonate, 10mM DTT) for 20 minutes at room temperature with shaking at
1000rpms. Iodoacetimide was added to 50mM, and samples were incubated in the dark for ten
minutes at room temperature with shaking at 1000rpms. 0.25ug of trypsin was added, and
samples were digested overnight at room temperature to ensure complete release of tryptic
peptides from the beads. Digested peptides were acidified with 10% TFA and stored on C18
StageTips for mass spectrometry analysis.
AP-MS/MS analysis
All chromatography was performed on an Easy-nLC 1000 (Thermo Scientific). Buffer A was
0.1% formic acid and Buffer B was 80% acetonitrile and 0.1% formic acid. LFQ samples
collected on the LTQ-Orbitrap QExactive were measured by developing a gradient from 9-32%
Buffer B for 94 minutes before washes at 50% then 95% Buffer B, for 120 minutes of total data
collection time. LFQ samples collected on the LTQ-Orbitrap Fusion Tribrid were measured by
developing a gradient from 9-32% Buffer B for 114 minutes before washes at 50% then 95%
Buffer B, for 140 minutes of total data collection time. xIP-MS samples were measured by
developing a gradient from 5-32% Buffer B for 214 minutes before washing with 60% then 95%
Buffer B, for 240 minutes of total data collection time. The flow rate was 250nl/min for all
SMC1A-GFP and XRCC6-GFP LFQ samples were measured on an LTQ-Orbitrap QExactive.
Full MS scans were collected from 300 to 1650 m/z with a resolution of 70,000 and an AGC
target of 3e6. MS/MS scans were collected in the orbitrap with a resolution of 17,500, an AGC
target of 1e5, an NCE of 25, and an intensity threshold of 8.3e2. TopN was set to 10, unassigned
and 1+ charged ions were excluded, and dynamic exclusion was set at 20 seconds.
xIP-MS: streamlined structural proteomics
PRPS1-GFP and MCM6-GFP LFQ samples were measured on an LTQ-Orbitrap Fusion Tribrid.
Full MS scans were collected from 400 to 1500 m/z with a resolution of 120,000 and an AGC
target of 4e5. MS/MS scans were collected in the linear ion trap using CID activation with a
resolution of 30,000, an AGC target of 1e4, collision energy of 35, and an intensity threshold of
5e3. Scans were collected in data-dependent top speed mode, ions of charge state 2-7+ were
considered, and dynamic exclusion was set at 60 seconds.
All xIP-MS samples were measured on an LTQ-Orbitrap-QExactive. Full MS scans were
collected from 300 to 1650 m/z with a resolution of 70,000 and an AGC target of 3e6. MS/MS
scans were collected in the orbitrap with a resolution of 17,500, an AGC target of 1e5, an NCE
of 25, and an intensity threshold of 4e2. TopN was set to 10, unassigned, 1+, and 2+ charged
ions were excluded, and dynamic exclusion was set at 20 seconds.
LFQ peptide identification and analysis
Thermo RAW files from LFQ AP-MS/MS measurements were analyzed with MaxQuant version using default settings and searching against the Uniprot curated human proteome (release
03/09/2014) [38, 39]. Cysteine carbamidomethyl was used as a fixed modification, and N-
terminal acetylation and methionine oxidation were used as variable modifications. Additional
options Match between runs, LFQ, and iBAQ were selected. Stoichiometry calculations and
volcano plots were produced essentially as described before using a one-way ANOVA test [22].
Statistical cutoffs were chosen such that no proteins were present as outliers on the control, non-
GFP side of the volcano plot.
xIP-MS: streamlined structural proteomics
xIP-MS cross-linked peptide identification and analysis
For cross-link identification, Thermo RAW files were converted to mgf format using MSConvert
with the peak picking option for levels “1- “ selected and the “Prefer Vendor” option checked
[40]. mgf files were analyzed in pLink version 1.21 with default settings to identify cross-linked
peptides with an FDR threshold of 0.05 using a precursor mass tolerance of 10ppm and a
fragment ion mass tolerance of 20ppm searching against specific interactors as identified by LFQ
analysis [30]. BS3 was used as the crosslinker, trypsin was used as the enzyme with a maximum
of two missed cleavages allowed, cysteine carbamidomethylation was included as a fixed
modification, and methionine oxidation was included as a variable modification. pLink
identifications were further filtered to include only matches with >=5 and <=40 residues per
peptide. Only cross-linked sites identified in two out of two independent replicates were
considered for further structural analysis (Supplemental Figure 1) [29]. Cross-link maps were
produced with xiNet, and distance constraints were analyzed with Xlink Analyzer in UCSF
Chimera [41-43]. All molecular visualizations were performed with UCSF Chimera [41].
Homology modeling
For PRPS1, only short internal gaps in the crystal structure were modelled using MODELLER in
UCSF chimera (PDB: 2h06) [31, 44]. All PSPS1 structural alignments were performed using
MatchMaker in UCSF Chimera [45].
All cohesin homology models were produced using SWISS-MODEL using alignments
calculated with EMBOSS Needle [46-48]. For SMC1A (PDB: 1w1w) and SMC3 (PDB: 4ux3)
N- and C-terminal head regions were aligned with their respective yeast PDB structure, and then
the SMC1A model was further aligned with a second SMC3 head domain present as a dimer in
xIP-MS: streamlined structural proteomics
the 4ux3 crystal structure for analysis of distance constraints. For comparative analysis using
prokaryote (PDB: 3zgx) and archea (PDB: 4i99) SMC homologs, a similar analysis was
performed. The cohesin hinge domain was similarly modeled from the mouse homolog (PDB:
Analysis of normal modes
Analysis of normal modes using anisotropic network analysis was conducted for XRCC5/6
(PDB: 1jeq) in Python with the prody package using default settings [49]. The twenty slowest
frequency modes were kept for visual analysis in VMD [50]. Figures were generated in prody,
and videos were made using VMD.
Data Access
The mass spectrometry RAW data, maxQuant identifications for LFQ analysis, and pLink
identifications for cross-linking analysis have been deposited to the ProteomeXchange
Consortium via the PRIDE partner repository with the dataset identifier PXD002987 [51].
xIP-MS: streamlined structural proteomics
High stringency washes effectively purify chromatin-associated complexes and permit chemical
cross-linking analysis
We first wanted to establish a protocol for effectively purifying stable protein complexes using a
simple, single affinity purification workflow. Our group previously optimized workflows for
high efficiency purification of GFP-tagged baits using a BAC transgene system [24-26]. Using
this method as a starting point, we tested whether more stringent washing conditions following
bead purification would be sufficient to enrich stable complexes with substantially increased
purity. After bead incubation in cell lysate buffer with 1% NP-40 and physiological salt, we
washed our beads twice with cell lysis buffer plus 1% NP-40 adjusted to 1M NaCl (Figure 1A).
This straightforward protocol effectively enriched a number of chromatin associated protein
complexes over background, on the order of hundreds of nanograms of purified material (Figure
2A, Supplemental Figure 1A-C). By gel and iBAQ analysis, we estimate this strategy isolates
stable, specific interactors with >95% purity. We were also able to efficiently cross-link purified
proteins on-beads directly following washing steps using the well-characterized BS3 cross-linker
in a borate-buffered saline buffer (Figure 2A).
Analysis of cross-linked peptides requires a database search that expands exponentially with the
number of peptides included. Such analysis becomes more computationally expensive and less
sensitive as the number of proteins included increases. Therefore, we used a standard targeted
method of defining a specific database of high-probability complex members for cross-link
searching. In our case, this involved identification of stable, specific interactors by LFQ analysis
[22, 39]. These high-confidence interactors were then included in our cross-link database for
targeted searching (Figure 1A). This approach is justified on two bases; first, cross-linked
xIP-MS: streamlined structural proteomics
peptides are already low-abundance in the pool of total peptides, therefore cross-linked peptides
from highly enriched, specific interactors are substantially more likely to be measured than those
from low-abundance, non-specific proteins. Second, we used a filtering strategy that effectively
removed spurious identifications to background peptides and specifically identified cross-linked
samples. We filtered out those cross-links where either the identified peptide was shorter than
five residues or longer than forty residues. This selectively removed matches where one long,
high scoring peptide compensated for a short, ambiguous partner in the scoring function. We
then additionally filtered out those cross-link identifications that were not observed in both
duplicate experiments, thus selectively removing remaining spurious matches (Supplemental
Figure 2A, Supplemental Figure 3A). To test the specificity of this filtering procedure, we
analyzed duplicate LFQ AP-MS/MS runs (without cross-linking) against a database of specific
LFQ interactors for each of five baits used in this study. These samples were highly enriched for
peptides of specific interactors, yet were not cross-linked and were thus negative controls. We
identified only two spurious cross-link matches across all five analyses using this filtering,
neither of which were identified as positive identifications when analyzing cross-linked samples,
strongly indicating this filtering effectively precludes false positives. We therefore concluded
that our high-stringency purification conditions obtained protein complexes with sufficient purity
to allow the confident identification of low abundance cross-linked peptides.
Structural benchmarking against the PRPP complex
We used PRPS1-GFP to structurally benchmark xIP-MS against the PRPP complex, a
multimeric enzyme associated with nucleotide synthesis, which has a known crystal structure
(PDB: 2h06) [31]. For the PRPP complex, we identified the bait protein, PRPS1, as well as two
xIP-MS: streamlined structural proteomics
associated proteins, PRPSAP1 and PRPSAP2, as significant interactors by LFQ analysis (Figure
3A). We then used iBAQ values from our LFQ experiment to calculate relative stoichiometries
for each subunit to the bait (Figure 3B). Using xIP-MS, we were able to identify 54 reproducible
cross-linked sites between these proteins (Figure 3C). Importantly, we were able to map all of
our unambiguous PRPS1 cross-linked sites onto the hexameric PRPS1 crystal structure within a
distance constraint of 34 Å (Figure 3D showing PRPS1 cross-links and Supplemental Figure 4A-
B showing all PRPS1 and PRPSAP1/2 cross-links). The distribution of identified cross-links
distances for PRPS1 was statistically distinct from the distribution of total distances and was thus
structurally informative (Figure 3E). This provides an important structural verification that xIP-
MS provides valid cross-link identifications.
A structural ensemble of PRPP complexes
PRPS1 has a canonical catalytically active hexameric form composed of three PRPS1 dimers.
The crystal structures of PRPSAP1 (PDB: 2c4k, RMSD: 0.901 Å with PRPS1) and PRPSAP2
(PDB: 2JI4, RMSD: 0.889 Å with PRPS1) are also known and indicate high structural homology
with PRPS1. PRPSAP1 and PRPSAP2 were observed at stoichiometries of 7-8% relative to
PRPS1, indicating either auxiliary subunit is present at slightly lower than one copy per two
PRPS1 hexamers (Figure 3B). We therefore considered whether our cross-linking data could
support the presence of an in vivo structural ensemble of PRPP complexes, where PRPSAP1 and
PRPSAP2 switch positions with canonical PRPS1 subunits within a single PRPP hexamer. This
possibility is particularly intriguing considering the reported role of PRPSAP1 in negative
regulation of PRPP catalytic function [52]. Their high structural homology enabled high-
confidence structural alignment of PRPSAP1 or PRPSAP2 with PRPS1 subunits, and we were
xIP-MS: streamlined structural proteomics
able to map all 51 of our unambiguous cross-linked sites onto either PRPS1, PRPS1-PRPSAP1
(6 inter-links), PRPS1-PRPSAP2 (2 inter-links), or PRPSAP1-PRPSAP2 (2 inter-links)
subcomplexes within a distance constraint of 34 Å (Supplemental Figure 4A-B). High structural
homology and our cross-linking data thus suggest an ensemble model of PRPP hexamers,
potentially offer a functional explanation for PRPSAP1’s negative regulatory function of PRPP,
and, again, indicate the structural validity of high confidence, reproducible xIP-MS cross-link
A model for cohesin head interactions
We next applied xIP-MS to the topological analysis of chromatin-associated protein complexes.
Cohesin is a dynamic and structurally intriguing protein complex involved in maintaining higher
order chromosome structure, most canonically during sister chromatid separation at mitosis. We
were able to effectively purify the core pentameric cohesin complex using SMC1A-GFP as a bait
(Figure 2A, Figure 4A). We purified SMC1A and SMC3 at essentially a 1:1 stoichiometric ratio,
consistent with their known dimeric interaction implicated in flexibly encircling chromosomes
(Figure 4B). However, we identified cohesin accessory proteins (RAD21, STAG1/2, PDS5B,
and WAPAL) at significantly lower stoichiometric ratios to the core cohesin dimer (Figure 4B).
This could indicate lower stoichiometric ratios in vivo, higher dynamism in bait interactions or
lower bait affinity, cell cycle effects, or sensitivity to washing conditions. We observed
numerous cross-links within the SMC1A and SMC3 head domains (Figure 4C). This data is
therefore complementary to previous studies, which both observed cross-linking predominantly
between the coiled-coils separating SMC1A and SMC3 head and hinge domains, potentially due
to different protein extraction methods or different cross-linking buffer conditions [11, 12]. We
xIP-MS: streamlined structural proteomics
conjectured that our cross-linking data would provide direct experimental support for a model of
the human SMC1A/SMC3 head interaction derived from available crystal structures. Therefore,
we modelled the human SMC1A and SMC3 head regions using crystal structures from their
yeast homologs (PDB: 1w1w and PDB: 4ux3, respectively) [53, 54]. We then aligned our
SMC1A model with a dimeric SMC3 head from the 4ux3 crystal structure (Figure 4D,
Supplemental Figure 5A). Similarly, we modelled the SMC1A and SMC3 hinge regions from
their respective chains in the mouse homolog (PDB: 2wd5) (Supplemental Figure 5B) [55]. In
sum, we found that 24/26 mappable cross-links were within a distance constraint of 34 Å using
these models, a number of which sat directly at the SMC1A/SMC3 head interface, indicating that
this straightforward modeling approach captures many architectural features of the human
cohesin head interaction (Figure 4D, Supplemental Figure 5C). Using a similar homology
modeling approach with prokaryote and archaea SMC homologs resulted in an increased number
of violations, therefore indicating eukaryotic cohesin structures represent the best available
model for the human complex (Supplemental Figure 6A) [56]. Therefore, xIP-MS provides
cross-linking data that complements previous XL-MS studies and directly supports a model of
the human cohesin head domain.
Conformational flexibility in XRCC5/6
Because xIP-MS is efficient enough to facilitate the use of replicates to identify very high-
confidence cross-linked sites, we sought to broaden the applications of xIP-MS by using it to
study potential conformational changes in flexible proteins. Such studies are typically difficult to
interpret because cross-links identified in flexible regions might exceed static spatial constraints
and therefore be disregarded as false. However, identification of such cross-links in multiple
xIP-MS: streamlined structural proteomics
independent experiments substantially increases confidence in their structural validity and points
towards conformational flexibility. Towards this end, we performed xIP-MS on the XRCC5/6
DNA damage repair complex [32]. XRCC5/6 has a flexible SAP domain thought to be involved
in conformational shifts involved in DNA recognition and binding at double-strand breaks
(DSBs) [32, 33]. Indeed, the SAP domain is resolved in the XRCC5/6 heterodimer without DNA
bound, though a presumably flexible linker region, residues 539-558, is not resolved (PDB:
1jeq). Moreover, the SAP domain is not resolved in the XRCC5/6 crystal structure with DNA
bound, further indicating the functional flexibility of this region in DNA contact (PDB: 1jey).
We were able to effectively purify the XRCC5/6 heterodimer at nearly 1:1 stoichiometry using
xIP-MS (Figure 2A, Figure 5A-B). We observed numerous cross-links within the XRCC5/6
heterodimer (Figure 5C). Although approximately 84% of our identified cross-linked seemed
spatially valid, we observed a few reproducible cross-linked sites that exceeded a spatial
constraint of even 40 Å (Supplemental Figure 7A-B). These cross-links we deemed excessive
violations, potentially indicative of conformational flexibility. Upon closer inspection, we
realized these cross-links generally correlate with regions of high B-factor in the crystal structure
and could be explained by two major conformation shifts (Figure 5D, Supplemental Figure 7A).
First, the DNA-binding Ku domain loop appears to possess substantial flexibility in the vertical
axis (Supplemental Figure 7A). This could be a consequence of cross-linking in solution in the
absence of DNA, and may represent native flexibility of the unbound dimer. We also observed
cross-links connecting the SAP domain with the Ku domain, indicating a conformational shift
where the flexible SAP domain approaches the Ku domain DNA binding ring. Analysis of
normal modes further supported substantial flexibility in the Ku and SAP domains
(Supplemental Figure 7C, Supplemental Video 1). Intriguingly, such conformational shifts could
xIP-MS: streamlined structural proteomics
be potentially indicative of allowed functional motion involved in stabilizing and capping DNA
DSB ends between the Ku and SAP domains (Figure 5D). Therefore, xIP-MS experiments
provide potential functional insight into conformational changes involved in XRCC5/6 DNA-
binding action. This highlights the ability of xIP-MS to provide high-confidence cross-link
identifications relevant in the study of protein flexibility and conformational states. Thus,
importantly, xIP-MS complements traditional crystallographic structural studies, which reveal
only static conformational states.
Topology of cell-cycle independent MCM subcomplex interactions
Because xIP-MS is a straightforward protocol requiring relatively small volumes of cell lysates,
we sought to show that xIP-MS could methodologically compare protein topologies under states
of perturbation. Traditionally, performing comparative cross-linking experiments has been
difficult due to the typical requirement of tens or hundreds of micrograms of protein at high
purity. However, we were easily able to perform xIP-MS experiments in duplicate qualitatively
comparing previously observed soluble MCM complexes in asynchronous and S-phase blocked
cells [37]. MCM is activated during DNA replication and loaded onto processive replication
forks; therefore, we expected MCM subcomplex topologies might change dramatically in
association with MCM activation during S-phase. However, we noted similar banding patterns
by gel analysis, similar interactors via LFQ proteomics, and similar relative stoichiometries for
interactors in both cell cycle states after MCM6-GFP purification (Figure 2A, Figure 6A-F,
Supplemental Figure 8A). Furthermore, we observed cross-linking between MCM2/4/6/7
subcomplexes (Figure 6B, 6D). Our gel analysis indicated three predominant cross-linked
species present in both states (Figure 2A). Our xIP-MS analysis showed a high degree of overlap
xIP-MS: streamlined structural proteomics
between cross-linked sites observed in both cell cycle states, indicating soluble MCM complexes
are topologically cell-cycle invariant (Figure 6F). Therefore, our data suggests a sequential
model of MCM subcomplex assembly, where ubiquitous soluble MCM subcomplexes are
assembled during loading onto processive replication forks during S-phase [34-36]. Our data also
suggests these soluble MCM subcomplexes may be in vast excess of active replication forks, as
they do not seem to be depleted from the soluble fraction during S-phase (Figure 2A). Our xIP-
MS data additionally provides structural insights into MCM subunit interfaces within these
subcomplexes. Finally, these experiments indicate the ease with which xIP-MS can be applied to
studying protein complex topologies under a variety of possible cellular or molecular
xIP-MS: streamlined structural proteomics
Chemical cross-linking coupled with mass spectrometry is becoming an increasingly popular
technique for studying large or difficult protein complex architectures and topologies at low
resolution. XL-MS is a particularly important tool for interrogating chromatin-associated protein
complexes, which are often highly dynamic, and subject to large conformational shifts when
enacting their functions. However, while affinity purification based strategies are commonly
coupled with mass spectrometry to identify specific protein interactions, typical XL-MS
workflows have traditionally demanded large (tens or hundreds of micrograms) amounts of pure,
homogenous complex coupled with time-consuming enrichment steps to facilitate detection of
cross-linked peptides over background. The difficulty of implementing and optimizing complex
cross-linking workflows has thus limited their widespread adoption. In general, XL-MS
workflows are scaling up towards proteome-wide studies often using cleavable cross-linkers, or
scaling down towards computational prediction of protein structures with high resolution using
broader-specificity or photo-activatable cross-linkers [16, 57]. We envision xIP-MS instead as a
workflow that could bridge the gap between proteome-wide and protein-specific XL-MS
workflows, offering peptide-resolution architectural and topological information with the ease of
coIP experiments particularly in the context of comparative cross-linking workflows [58-61].
This study establishes xIP-MS as an additional tool for molecular and structural biologists,
offering a flexible and robust methodological platform adaptable to a variety of diverse
It is worth noting that the xIP-MS protocol as presented is designed to enrich stable core
complexes with high efficiency and purity from relatively small lysate volumes. The high
stringency washing steps necessitated by this workflow can preclude highly dynamic or transient
xIP-MS: streamlined structural proteomics
interactions between even stoichiometric interactors. Similarly, salt sensitive interactions may be
impeded by xIP-MS. As a general rule, specific purification conditions may be required for
different complexes; this should be taken into consideration for the complex in question.
However, the workflow we present in this study represents a generally effective starting point, as
we have shown by applying xIP-MS successfully to a number of different protein complexes
with different downstream applications, including: heterogeneous assemblies, homology
modeling, structural dynamics, and comparative cross-linking. xIP-MS therefore represents a
simplified and flexible alternatative to existing on-bead cross-linking methodologies.
Although on-bead cross-linking workflows offer many experimental benefits, one notable
drawback to on-bead cross-linking workflows is the potential to cross-link purified proteins to
the bead itself, potentially facilitating misidentifications. Here, we use a small GFP nanobody
(12kDa) with stringent data filtering to decrease the likelihood of spurious identifications as
much as possible. However, alternate strategies include Ni-NTA purification for His-tagged
proteins as Ni-NTA is a non-amine containing substrate, using a membrane permeable cross-
linker prior to cell lysis and purification, or using engineered high-affinity, lysine-free
nanobodies [15, 16, 27]. In general, careful experimental design and data analysis should
minimize the risk of false positive identifications from on-bead cross-linking workflows.
xIP-MS also offers much promise as a functional follow up to more traditional structural
workflows. Because xIP-MS is sufficiently straightforward to allow multiple replicates, high
confidence, reproducible cross-linking with xIP-MS can facilitate structural dynamics analysis as
we have shown for XRCC5/6. Importantly, perturbation experiments now become substantially
streamlined, indicated by our analysis of cell cycle invariance in soluble MCM subcomplexes.
Quantitative cross-linking with isotopic linkers offers great potential for comparing
xIP-MS: streamlined structural proteomics
conformational states under perturbations, and it is possible xIP-MS could contribute towards
further developments in quantitative cross-linking workflows [58-61].
It is likely developments in protein purification reagents, novel cross-linking chemistries and
softwares, and faster, more sensitive instruments will improve xIP-MS workflows in the future.
For example, xIP-MS would benefit directly from novel ultrahigh-affinity GFP nanobody
dimers, as reported recently [62]. Also, developments in enrichable or cleavable cross-linkers
could facilitate higher cross-link identification rates [16, 20, 63]. Therefore, xIP-MS can also be
considered as an experimental platform for rapid optimization of novel cross-linking strategies,
thereby extending the toolbox of structural proteomics technologies.
M.M. and M.V. received funding for this project from the Marie Curie FP7 DevCom ITN.
xIP-MS: streamlined structural proteomics
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xIP-MS: streamlined structural proteomics
Figure Legends
Figure 1: xIP-MS: a workflow for the analysis of protein complex topologies
Figure 2: High stringency washes effectively purify protein complexes for on-bead cross-linking
A) BS3- lanes represent one half of an xIP-MS experiment using 1mL of cell lysate. The
second half was resuspended in cross-linking buffer for on-bead cross-linking as
described in text, and is shown in the BS3+ lanes. All lanes were resolved on the same
gel at the same exposure; black lines indicated where lanes were cropped together for
Figure 3: xIP-MS analysis of PRPP reveals high confidence distance constraints
A) LFQ analysis of PRPS1 was performed with triplicate pulldowns. Outliers are indicated
in red, and background binders are indicated in black. Outlier cutoffs were drawn such
that no proteins were present in the non-GFP quadrant on the volcano plot. B)
Stoichiometry analysis was performed as decribed. All ratios are calculated by setting the
bait (PRPS1) equal to 1. Error bars indicate standard deviations from triplicate samples.
C) Identified cross-links for PRPP. The cross-link map was drawn in xiNet. Purple lines
indicate self-links, and green lines indicate inter-protein cross-links. Ambiguous cross-
links (the same peptide in multiple proteins) are indicated by dashed lines. Homotypic
cross-links (the same peptide cross-linked to itself, indicating multimerization) are drawn
in red. Uniprot annotated domains are colored variously along the protein sequence bar.
D) PRPS1 cross-links mapped onto the PDB: 2h06 crystal structure. Cross-links under 34
Å are colored in blue. PRPS1 monomers are colored in light grey. The silhouette of the
PRPS1 holo-hexamer surface is shown in transparent grey.
xIP-MS: streamlined structural proteomics
Figure 4: xIP-MS suggests a model for human cohesin head interactions
A) LFQ analysis identifies canonical cohesin tetrameric subunits as interactors of SMC1A.
Data is plotted as described previously. B) SMC1A and SMC3 interact at a near 1:1
stoichiometry as indicated by iBAQ values. C) Cross-links between SMC1A and SMC3
preferentially localize towards the head domains, indicated in green on the protein
sequence bars. D) Homology modeling allows mapping of SMC1A and SMC3 cross-
links onto models for the human proteins. SMC1A is colored light grey, and SMC3 is
colored in dark grey. Cross-links below 34 Å are shown in blue, and cross-links above 34
Å are shown in red. The silhouette represents the surface of the SMC3 dimer from the
PDB: 4ux3 crystal structure used for aligning the SMC1A and SMC3 models.
Figure 5: xIP-MS reveals conformational changes in XRCC5/6
A) LFQ identifies XRCC5/6 as robust interactors, in agreement with our gel analysis. B)
XRCC5/6 stoichiometry is nearly 1:1 as indicated by iBAQ values. C) Cross-linking
within the XRCC5/6 heterodimer. The cross-link map is colored as described previously.
D) XRCC5/6 are colored by B-factor as indicated on the key; the protein backbone radii
are scaled similarly by size. Cross-links within 40 Å are shown in blue, and cross-links
longer than this threshold are shown in red. Major conformational shifts explaining
observed cross-linking patterns are displayed as yellow arrows. The structure of PDB:
1jeq is used to display cross-links as the SAP domain is resolved only in this structure;
however, the surface of the DNA structure bound to XRCC5/6 from PDB: 1jey is
displayed in transparent yellow, after alignment with 1jeq using MatchMaker in UCSF
xIP-MS: streamlined structural proteomics
Figure 6: xIP-MS reveals S-phase independent MCM subcomplex topologies
A) MCM2/4/6/7, MCM10, and MCMBP are observed as interactors with MCM6 in
asynchronous cells. B) Cross-linking analysis reveals inter-protein contacts within
soluble MCM complexes in asynchronous cells. C) Similarly, MCM2/4/6/7, MCM10,
and MCMBP are observed as interactors with MCM6 in S-phase blocked cells. D)
Cross-linking analysis reveals a similar topology in soluble S-phase MCM complexes. E)
Relative stoichiometries to the bait as indicated by iBAQ values show MCM subunit
stoichiometries do not change substantially when cells are blocked in S-phase. F) Venn
diagram indicates substantial overlap of cross-linked sites between MCM subunits after
S-phase block.
Bait Protein
Collect whole cell
lysates with
singly-tagged bait
Perform single-AP
with high stringency
Identify specific
interactors via LFQ
On-bead cross-linking
and sample preparation
Identify cross-linked
peptides from specific
Identify relative subunit
Figure 1
MCM6-GFP Asynchronous
MCM6-GFP S-phase
BS3 - + - + - + - + - +
Figure 2
GFP/Non-GFP Ratio
iBAQ Ratio to PRPS1
Figure 3
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
Xlink Count
Xlink Distance (Å)
PRPP Theoretical Xlinks (All)
PRPP Observed Xlinks
Welch's T-test p -value: 2.09x10
GFP/Non-GFP Ratio
iBAQ Ratio to SMC1A
Figure 4
GFP/Non-GFP Ratio
iBAQ Ratio to XRCC6
Figure 5
Ku Domain
SAP Domain
iBAQ Ratio to MCM6
GFP/Non-GFP Ratio
GFP/Non-GFP Ratio
Figure 6
... Adaptation of AP-MS to study protein interactions at the plasma membrane would typically require stabilizing protein interactions, using crosslinking, for instance (8,9), which can elongate the process and convolute data interpretation, or require the systematic exploration of collections of detergents and lysis conditions that optimize recovery of specific complexes (10,11). Additionally, blue native gels have been used to identify membrane protein complexes (12,13) but require pre-solubilization of membrane proteins in mild lysis conditions to maintain complexes, presenting a similar limitation to affinity purification in studying proteins of hydrophobic nature. ...
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Plasma membrane proteins are critical mediators of cell-cell and cell-environment interactions, pivotal in intracellular signal transmission vital for cellular functionality. Proximity-dependent biotinylation approaches such as BioID combined with mass spectrometry have begun illuminating the landscape of proximal protein interactions within intracellular compartments. However, their deployment in studies of the extracellular environment remains scarce. Here, we present extracellular TurboID (ecTurboID), a method designed to profile cell surface interactions in living cells on short timescales. We first report on the careful optimization of experimental and data analysis strategies that enable the capture of extracellular protein interaction information. Leveraging the ecTurboID technique, we unveiled the proximal interactome of multiple plasma membrane proteins, notably the epidermal growth factor receptor (EGFR). This led to identifying the low-density lipoprotein receptor (LDLR) as a newfound extracellular protein associating with EGFR, contingent upon the presence of the EGF ligand. We showed that 15 minutes of EGF stimulation induced LDLR localization to the plasma membrane to associate with proteins involved in EGFR regulation. This modified proximity labelling methodology allows us to dynamically study the associations between plasma membrane proteins in the extracellular environment. One Sentence Summary We developed extracellular TurboID (ecTurboID) as a new proximity dependent biotinylation approach that can capture dynamic interactions at the cell surface, identifying Low-Density Lipoprotein Receptor as a new ligand-dependent extracellular partner of Epidermal Growth Factor Receptor.
... Quantification labels such as TMT labels offer multiplexing across a variety of conditions (Li et al., 2021). However, AP-MS does not offer any insights into the binding sites for the interactors (Makowski et al., 2016). Recent advancements in chemo-proteomics resolve interactors in different protein regions by leveraging site-specific unnatural amino acid incorporation of photo-chemical crosslinking 4 reagents to covalently crosslink interactors (Futran et al., 2015;Shah et al., 2020). ...
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Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.
... Therefore, all cross-links detailed in this study refer to intra-protein cross-links (intra-links) of Pgp or the antibodies. The quantity of the identified cross-links from the living cell or on-bead approaches is at the same order of magnitude as that previously reported in similar studies [41,42]. Membrane proteins being difficult to handle, the number of identified cross-links in the present study is considered efficient enough and some functional conclusions could be drawn. ...
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The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp's conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp's solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskele-ton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.
... The basic approach involves chemically installing a bifunctional reagent between two reactive amino acid residues and detecting the linkage points using methods derived from bottom-up proteomics 4,5 . XL-MS can be used to model protein structure [6][7][8] , validate local interactome organization when paired with affinity isolation [9][10][11] , and even the map entire cellular networks [12][13][14] . Although the major elements of the technique have a long history, its application to whole cells for structural analysis is relatively recent, enabled primarily by higher sensitivity mass spectrometers and computational methods that can locate these direct linkage sites in highly complex digests. ...
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Crosslinking mass spectrometry (XL-MS) is a valuable technique for the generation of point-to-point distance measurements in protein space. Applications involving in situ chemical crosslinking have created the possibility of mapping whole protein interactomes with high spatial resolution. However, an XL-MS experiment carried out directly on cells requires highly efficient software that can detect crosslinked peptides with sensitivity and controlled error rates. Many algorithmic approaches invoke a filtering strategy designed to reduce the size of the database prior to mounting a search for crosslinks, but concern has been expressed over the possibility of reduced sensitivity with such strategies. Here we present a full upgrade to CRIMP, the crosslinking app in the Mass Spec Studio, which implements a new strategy for the detection of both component peptides in the MS2 spectrum. Using several published datasets, we demonstrate that this pre-searching method is sensitive and fast, permitting whole proteome searches on a conventional desktop computer for both cleavable and noncleavable crosslinkers. We introduce a new strategy for scoring crosslinks, adapted from computer vision algorithms, that properly resolves conflicting XL hits from other crosslinking reaction products, and we present a method for enhancing the detection of protein-protein interactions that relies upon compositional data.
Comprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS). By this method, in vivo cross-linking was introduced to covalently fix intracellular PPIs in their functional states to assure all PPIs could be integrally maintained during cell disruption. In addition, the chemically cleavable crosslinkers which were employed enabled unbinding of PPIs for in-depth identification of components within the interactome and biological analysis, while allowing binding of PPIs for cross-linking-mass spectrometry (CXMS)-based direct interaction determination. Multi-level information on targeted PPIs network can be obtained by ICAP-MS, including composition of interacting proteins, as well as direct interacting partners and binding sites. As a proof of concept, the interactome of MAPK3 from 293A cells was profiled with 6.15-fold improvement in identification than by conventional AP-MS. Meanwhile, 184 cross-link site pairs of these PPIs were experimentally identified by CXMS. Furthermore, ICAP-MS was applied in the temporal profiling of MAPK3 interactions under activation by cAMP-mediated pathway. The regulatory manner of MAPK pathways was presented through the quantitative changes of MAPK3 and its interacting proteins at different time points after activation. Therefore, all reported results demonstrated that the ICAP-MS approach may provide comprehensive information on interactome of targeted protein for functional exploration.
Background: Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. However, up to date most of the studies utilising XL-MS to characterise individual protein complexes’ topology have been carried out on over-expressed or recombinant proteins, which might not accurately represent native cellular conditions. Methods: We performed XL-MS using MS-cleavable crosslinker disuccinimidyl sulfoxide (DSSO) after immunoprecipitation of endogenous BRG/Brahma-associated factors (BAF) complex and co-purifying proteins. Data are available via ProteomeXchange with identifier PXD027611. Results: Although we did not detect the expected enrichment of crosslinks within the BAF complex, we identified numerous crosslinks between three co-purifying proteins, namely Thrap3, Bclaf1 and Erh. Thrap3 and Bclaf1 are mostly disordered proteins for which no 3D structure is available. The XL data allowed us to map interaction surfaces on these proteins, which overlap with the non-disordered portions of both proteins. The identified XLs are in agreement with homology-modelled structures suggesting that the interaction surfaces are globular. Conclusions: Our data shows that MS-cleavable crosslinker DSSO can be used to characterise in detail the topology and interaction surfaces of endogenous protein complexes without the need for overexpression. We demonstrate that Bclaf1, Erh and Thrap3 interact closely with each other, suggesting they might form a novel complex, hereby referred to as TEB complex. This data can be exploited for modelling protein-protein docking to characterise the three-dimensional structure of the complex. Endogenous XL-MS might be challenging due to crosslinker accessibility, protein complex abundance or isolation efficiency, and require further optimisation for some complexes like the BAF complex to detect a substantial number of crosslinks.
Recent advancements in mass spectrometry (MS) now enable all levels of protein structures to be characterized, including primary protein sequence, post-translational modifications, and three-dimensional protein conformations. However, protein conformational studies by MS require the use of many separate techniques that are performed independently of each other. Herein, we described a contained-electrospray (ES) experiment that has potential to integrate peptide/protein cross-linking with the general MS workflow. In our experiment, cross-linking of protein/peptide occurs simultaneously with ionization after analytes, and cross-linkers are sprayed from two separate ES emitters. The online cross-linking process occurring in the charged microdroplet environment was optimized using trilysine peptide and bis(sulfosuccinimidyl)suberate cross-linker. We detected the electrostatic complex between analyte and cross-linker, the mono-linked intermediate, and the fully cross-linked product, allowing us to correctly predict the sequence of reaction events in the cross-linking process. Importantly, we observed that the terminal fully cross-linked product is composed of two distinct conformations. In one form, the product involved cross-linking between two ε-NH2 amines in lysine residues, while the other conformer was formed by a reaction between one ε-NH2 amine and the N-terminus. The experimental conditions for selecting one cross-linked species over others during the online ES ionization-MS analysis have been detailed. Appropriate parameters enabled the reaction between α-lactalbumin proteins and cross-linkers using a non-denaturing spray condition. These results establish a framework for a future development in high-throughput structural MS method, where all levels of protein information can be gathered in a single experiment.
Crosslinking mass spectrometry (crosslinking-MS) is a versatile tool providing structural insights into protein conformation and protein-protein interactions. Its medium-resolution residue-residue distance restraints have been used to validate protein structures proposed by other methods and have helped derive models of protein complexes by integrative structural biology approaches. The use of crosslinking-MS in integrative approaches is underpinned by progress in estimating error rates in crosslinking-MS data and in combining these data with other information. The flexible and high-throughput nature of crosslinking-MS has allowed it to complement the ongoing resolution revolution in electron microscopy by providing system-wide residue-residue distance restraints, especially for flexible regions or systems. Here, we review how crosslinking-MS information has been leveraged in structural model validation and integrative modeling. Crosslinking-MS has also been a key technology for cell biology studies and structural systems biology where, in conjunction with cryoelectron tomography, it can provide structural and mechanistic insights directly in situ.
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Human Ku70 is a well-known endogenous nuclear protein involved in the non-homologous end joining pathway to repair double-stranded breaks in DNA. However, Ku70 has been studied in multiple contexts and grown into a multifunctional protein. In addition to the extensive functional study of Ku70 in DNA repair process, many studies have emphasized the role of Ku70 in various other cellular processes, including apoptosis, aging, and HIV replication. In this review, we focus on discussing the role of Ku70 in inducing interferons and proinflammatory cytokines as a cytosolic DNA sensor. We explored the unique structure of Ku70 binding with DNA; illustrated, with evidence, how Ku70, as a nuclear protein, responds to extracellular DNA stimulation; and summarized the mechanisms of the Ku70-involved innate immune response pathway. Finally, we discussed several new strategies to modulate Ku70-mediated innate immune response and highlighted some potential physiological insights based on the role of Ku70 in innate immunity.
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It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.
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Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low resolution of cross-linking has so far precluded its use in determining structures de novo. Cross-linking resolution has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high-resolution photo-cross-linking with conformational space search to investigate the structure of human serum albumin (HSA) domains, from purified samples, and in it's native environment, human blood serum. Our approach is able to determine HSA domain structures with good accuracy: RMSD to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8 Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on HSA demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.
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SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organization. SMC-containing complexes are large (more than 650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here, we have used amino acid-selective cross-linking and mass spectrometry combined with structure prediction to develop a full-length molecular draft three-dimensional structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularities. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.
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Structural characterization of large multi-subunit protein complexes often requires integrating various experimental techniques. Cross-linking mass spectrometry (XL-MS) identifies proximal protein residues and thus is increasingly used to map protein interactions and determine the relative orientation of subunits within the structure of protein complexes. To fully adapt XL-MS as a structure characterization technique, we developed Xlink Analyzer, a software tool for visualization and analysis of XL-MS data in the context of the three-dimensional structures. Xlink Analyzer enables automatic visualization of cross-links, identifies cross-links violating spatial restraints, calculates violation statistics, maps chemically modified surfaces, and allows interactive manipulations that facilitate analysis of XL-MS data and aid designing new experiments. We demonstrate these features by mapping interaction sites within RNA polymerase I and the Rvb1/2 complex. Xlink Analyzer is implemented as a plugin to UCSF Chimera, a standard structural biology software tool, and thus enables seamless integration of XL-MS data with, e.g. fitting of X-ray structures to EM maps. Xlink Analyzer is available for download at Copyright © 2015. Published by Elsevier Inc.
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xiNET is a visualization tool for exploring cross-linking/mass spectrometry results. The interactive maps of the cross-link network that it generates are an extended type of node-link diagram. In these maps xiNET displays: (i) residue resolution positional information including linkage sites and linked peptides; (ii) ambiguous results; (iii) additional sequence information such as domains; and (iv) all types of cross-linking reaction product. xiNET runs in a browser and exports vector graphics which can be edited in common drawing packages to create publication quality figures. Availability: xiNET is open source, released under the Apache version 2 license. Results can be viewed by uploading data to or by downloading the software from and running it locally. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
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Through their association with a kleisin subunit (Scc1), cohesin’s Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3’s adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin’s association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin’s dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)-tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.
Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines, pLink and xQuest. The identified inter-linked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes. This article is protected by copyright. All rights reserved.
Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines, pLink and xQuest. The identified inter-linked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes. This article is protected by copyright. All rights reserved. © 2015 The Protein Society.
Chromosome segregation depends on sister chromatid cohesion mediated by cohesin. The cohesin subunits Smc1, Smc3, and Scc1 form tripartite rings that are thought to open at distinct sites to allow entry and exit of DNA. However, direct evidence for the existence of open forms of cohesin is lacking. We found that cohesin's proposed DNA exit gate is formed by interactions between Scc1 and the coiled-coil region of Smc3. Mutation of this interface abolished cohesin's ability to stably associate with chromatin and to mediate cohesion. Electron microscopy revealed that weakening of the Smc3-Scc1 interface resulted in opening of cohesin rings, as did proteolytic cleavage of Scc1. These open forms may resemble intermediate states of cohesin normally generated by the release factor Wapl and the protease separase, respectively. Copyright © 2014, American Association for the Advancement of Science.