Enrichment of Cross-Linked Peptides Using Charge-Based Fractional

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Enrichment of cross-linked peptides using charge based fractional diagonal chromatography (ChaFRADIC) Verena Tinnefeld, Saskia Venne, Albert Sickmann, and René Peiman Zahedi J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00587 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 13, 2016

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Journal of Proteome Research

Enrichment of cross-linked peptides using charge based fractional diagonal chromatography (ChaFRADIC)

Verena Tinnefeld1, Saskia Venne1, Albert Sickmann1,2,3, René P. Zahedi1,* 1

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany

2

Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen

UK 3

Medizinisches Proteom Center, Ruhr Universität Bochum, Bochum, Germany

* Corresponding author: Dr. René P. Zahedi Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V. Otto-Hahn-Str. 6b D-44227 Dortmund Germany Tel.: +49 (0) 231-1392-4143 Fax: +49 (0) 231-1392-4850 E-mail: [email protected]

KEYWORDS Protein cross-linking, enrichment, strong cation exchange chromatography, ChaFRADIC, mass spectrometry

1

ACS Paragon Plus Environment


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Abstract Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of crosslinking products their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. Here, we present here a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Based on two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture and crosslinked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible and independent of the cross-linking chemistry and cross-linker properties.

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Introduction Chemical cross-linking of proteins combined with mass spectrometry has become a widely used method to investigate the structure of proteins and protein complexes1. X-ray crystallography and NMR spectroscopy are still the most important methods for high resolution elucidation of protein and protein complex structures. However, protein cross-linking mass spectrometry (XL-MS) is a comparatively simple method, which can substantially support these classical methods with low resolution information2, 3. In contrast to X-ray crystallography there is no need to crystallize proteins and XL-MS requires lower sample amounts than NMR. As XL-MS is mainly combined with proteolytic digestion in so-called bottom-up approaches, there are virtually no limitations to protein/complex size, which is limited in NMR analysis. In XL-MS proteins are treated in their native states with a bifunctional chemical compound of specific length that is reactive against certain amino acid side chains or functional groups, mostly primary amines. If respective amino acid side chains have the appropriate inter- or intramolecular distance they can be covalently linked, which preserves their relative positions4. Afterwards, cross-linked proteins are enzymatically digested, followed by LCMS and data analysis using specific software tools5 to identify the amino acids which have been crosslinked. Currently, the two biggest challenges protein cross-linking mass spectrometry faces are: (i) data analysis, due to the complexity of cross-linked peptide MS/MS spectra, as well as (ii) the low abundance of cross-linked peptides in complex samples. On the one hand, fragment ion spectra of cross-linked peptides contain fragments from two connected peptides instead of one, which makes them inaccessible to classical data base search engines. To address this problem, dedicated software tools for data analysis of cross-linking experiments have been developed. Although more than 20 programs exist, new software tools are constantly developed6, 7 – this might be partially attributed to the complexity of the different cross-linking chemistries available, but also to a still existing lack of standardization and thus resulting individual solutions. On the other hand, cross-linked peptides are low abundant in typical experiments. First, the number of amino acid side chains within a protein or protein complex (i) with appropriate functional groups (ii) at the appropriate molecular distance, (iii) 3



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which are well-accessible in solution, is limited. Second, even for suitable residues the cross-linking reaction often has a relatively low efficiency. Thus, as for post-translationally modified peptides such as phosphopeptides8, there is a need for methods that allow a specific enrichment of underrepresented cross-linked peptides from the bulk of non-modified peptides. So far there are only few strategies for enriching cross-linked peptides9. One possibility is the crude enrichment via size exclusion chromatography (SEC), as cross-linked peptides comprised of two fully-cleaved peptides are on average larger than non-linked peptides9-11. Another approach makes use of a cross-linking reagent with a biotin moiety for enrichment of cross-linked peptides12,13. Although elegant, this approach cannot exclude the co-enrichment of mono-links. Furthermore, strong cation exchange chromatography (SCX) can be used for enrichment of cross-linked peptides that on average have higher charges states than non-linked peptides, as demonstrated by Fritzsche et al.14 The potential of SCX to study protein modifications with low stoichiometry has been demonstrated before15. In contrast, Buncherd et al. developed a two-dimensional SCX approach, which makes use of a chemically cleavable cross-linking reagent16. After a first dimension SCX, cross-links are cleaved into linear peptides thus inducing a shift to earlier retention times in a second dimension SCX. However, as the cross-linked peptides are cleaved, the information which peptides were linked is lost. Thus, based on the cleaved peptides identified after enrichment, theoretical masses of putative intact cross-linked peptides are calculated and compared to an LC-MS analysis of the intact sample without enrichment, rendering the whole procedure laborious. Here, we present a novel and universal approach for the enrichment of cross-linked peptides that is independent of the cross-linking chemistry. Our strategy is based on Charge-based Fractional Diagonal Chromatography (ChaFRADIC) which we originally developed to enrich N-terminal peptides17. There, free primary amines (N-termini, Lys residues) are blocked on the protein level with dimethyl. After digestion with trypsin (having an ArgC specificity due to blocked Lys residues) peptides are separated by SCX at pH 2.7 into fractions with theoretical net charge states of +1, +2, +3, and +4. Afterwards, individual fractions are trideutero-acetylated, such that free N-termini generated upon the proteolytic digestion will be blocked. Thus, in pH 2.7 SCX the net charge of internal peptides 4


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will be reduced by one, whereas N-terminal peptides which were already dimethylated or endogenously acetylated retain their net charge. Consequently, in a second dimension SCX, internal peptides shift to earlier fractions, while N-terminal peptides elute in the same as before. In XL-ChaFRADIC (workflow depicted in figure 1), we use LysC as proteolytic enzyme to increase the theoretical net charge distance between non-linked and cross-linked peptides, compared to a conventional trypsin digestion. Then, peptides with high net charge states are collected in a first dimension of SCX. To remove co-enriched non-linked peptides with multiple charged residues and make cross-linked peptides accessible for LC-MS analysis, we digest the collected samples in a second step with trypsin, followed by a second dimension of SCX in which fractions containing highly charged peptides were collected.

This approach is universally applicable to all kinds of cross-linkers, irrespective of the applied chemistry and whether they are (i) non-cleavable, (ii) MS or chemically cleavable, (iii) chemical or photo-reactive. We demonstrate, that our new approach allows effective enrichment of cross-linked peptides from samples of low (pure cross-linked BSA, pBSA), medium (cross-linked BSA mixed with 5 proteins, mBSA) and high (cross-linked BSA in HeLa lysate, hBSA) complexity.

Materials and methods Materials and reagents Ammonium bicarbonate (ABC), bovine serum albumin (BSA), bovine hemoglobin, β-lactoglobulin, ovalbumin, lysozyme, magnesium chloride (MgCl2), guanidine hydrochloride (GuHCl) and iodoacetamide (IAA) were purchased from Sigma Aldrich (Steinheim, Germany). Dithiothreitol (DTT) and complete Mini EDTA fee were acquired from Roche Diagnostics (Mannheim, Germany). Tris-(hydroxymethyl)-aminomethane

(Tris),

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid

(HEPES) and sodium dodecyl sulfate (SDS) were purchased from Applichem (Darmstadt, Germany) and sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen sulfate (KH2PO3), 5



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hydrochlorid acid, sulfonic acid, benzonase, ethanol and ethylenediaminetetraacetic acid (EDTA) from Merck (Darmstadt, Germany). Bis(sulfosuccinimidyl) 2,2,7,7-suberate-d0/4 (BS3-d0/4), bicinchoninic acid (BCA) assay and BioBasic-4 C4 bulk material were acquired from Thermo Scientific (Schwerte, Germany). Oligo R3 bulk media was purchased from life technologies (Darmstadt, Germany). SPEC Pt C18Ar desalting tips were purchased from Agilent (Waldbronn, Germany). r-Lys-C, MS grade, and trypsin Gold, MS grade, were purchased from Promega (Mannheim, Germany). PD Spin Trap G-25 microcolumns were purchased from GE Healthcare (Freiburg, Germany). All UPLC-grade solvents were obtained from Biosolve (Valkenswaard, Netherlands).

Preparation of cross-linked BSA in samples of low, medium and high complexity For the cross-linking reaction BSA was dissolved in 20 mM HEPES, pH 7.2 at 1 mg/mL. BS3-d0/4 was dissolved in DMSO (25 mM). A 1 M NH4HCO3 solution was prepared for quenching. BS3-d0/4 was added to the BSA solution in 10-fold molar excess and incubated for 30 min at room temperature. The reaction was quenched for 10 min with NH4HCO3 in 10-fold molar excess of the cross-linking reagent. For the pure cross-linked BSA (pBSA sample), the solution was dried completely after quenching. For the protein background (mBSA sample), non-cross-linked proteins (bovine hemoglobin, BSA, βlactoglobulin, lysozyme and ovalbumin) were dissolved at 3 mg/mL in water and mixed at equal amounts (1:1:1:1:1, w/w). Cross-linked BSA was mixed with this protein solution in a ratio of 1:6 (w/w) and the mixture was dried completely. For the workflow with HeLa background (hBSA), HeLa cell pellets were lysed in 50 mM Tris-HCl, 150 mM NaCl, 1% SDS at pH 7.8 and complete Mini EDTA free. 150 U Benzonase and 2 mM MgCl2 were added and incubated at 37 °C for 30 min. The lysate was centrifuged at 4 °C, 18,000 g for 30 min and the supernatant was collected. Protein concentration was determined using the BCA assay. Afterwards SDS was removed by ethanol precipitation. Ice cold ethanol was added (1:9, v/v) to the sample and incubated for 2 hours at -40 °C. The sample was centrifuged at 18,000 g for 30 min (4 °C). The supernatant was discarded; the protein pellet was dried in a vacuum centrifuge and afterwards 6


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dissolved in 4 M guanidine hydrochloride (GuHCl). Cross-Linked BSA was mixed in the ratio 1:15 (w/w) with HeLa lysate and dried completely. First proteolytic digestion with Lys-C Dried samples were dissolved in 4 M GuHCl. Cysteines were reduced with 10 mM DTT for 30 min at 56 °C. Afterwards, free sulfhydryl groups were alkylated using 20 mM IAA for 30 min at RT in the dark. Samples were diluted to final concentration of 0.2 M GuHCl with 25 mM Tris-HCl, 1 mM EDTA, pH 8.5. Lys-C was added in a 1:20 (w/w) ratio and incubated overnight at 37 °C. Digestion was quenched with 1% TFA (final concentration). Digestion efficiency was controlled by monolithic RP separation as described previously18. After Lys-C digestion, samples were desalted. For the pBSA sample self-made microcolumns with BioBasic-4 C4 particles were used19. These were prepared in a pipette tip with Whatman paper as frit and a 250 µg BioBasic4-bed. The mBSA and hBSA samples were desalted using commercial C18 Spec tips. For all desalting steps, the procedure was the same: Conditioning with 60% acetonitrile/0.1% TFA, equibrilation twice with 0.1% TFA. The sample was loaded twice and washed twice with 0.1% TFA. For elution 60% acetonitrile/0.1% TFA, followed by 100% acetonitrile were applied. Microcolumns were used in a centrifuge, all steps besides sample loading and elution at 1000 g for 2 min; sample loading and elution at 600 g for 5 min. Spec tips were used with a vacuum station at 5 inHg. Desalted samples were dried completely and dissolved in 15 µL (pBSA, 6 ug) or 50 µL (mBSA/hBSA, 100 µg) of SCX buffer A (10 mM KH2PO4, 20% acetonitrile, pH 2.7).

1st Dimension Enrichment of crosslinked peptides 1st dimension SCX separations were performed using a U3000 HPLC system (Thermo Scientific) and a 150 × 1 mm (for pBSA samples) or a 150 x 0.3 mm (for mBSA and hBSA samples) POLYSULFOETHYL A column (PolyLC INC, Columbia, US, 5 µm particle size, 200 Å pore size). A tertiary buffer system was used: SCX buffer A (10 mM KH2PO4, 20% acetonitrile, pH 2.7), SCX buffer B (10 mM KH2PO4, 250 mM KCl, 20% acetonitrile, pH 2.7), and SCX buffer C (10 mM KH2PO4, 600 mM NaCl,20% acetonitrile, pH 2.7). 7



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6 µg of the pBSA sample were separated with the smaller column (150 x 0.3 mm) at a flow rate of 5 µL/min. The optimized gradient was as follows: 100% A for 15 min, followed by a linear increase of B from 0% to 15% in 10 min. B was kept at 15% for 10 min and then further increased linearly to 30% in 10 min. 30% B was kept for 10 min, followed by a linear increase of B to 100% in 1 min. B was kept at 100% for 9 min. Afterwards C was set to 100% for 9 min. The gradient allowed a separation of peptides according to their theoretical net charge states at pH 2.717. Five Fractions representing theoretical peptide net charge states +1, +2, +3, +4, > +4 were collected manually. The mBSA and hBSA samples (100 µg each) were separated with the 150 µm ID column. The optimized gradient was as follows: 100% A for 10 min, followed by a linear increase of B from 0% to 15% in 9.3 min. B was kept at 15% for 8.7 min and then further increased linearly to 30% in 8 min. 30% B was kept for 11 min, followed by a linear increase of B to 100% in 5 min. B was kept at 100% for 5 min. Afterwards C was set to 100% for 5 min. Five fractions according to the expected peptide net charge states +1, +2, +3, +4, > +4 were collected automatically using a fraction collection option. All collected fractions were dried in a vacuum centrifuge and desalted with C4 micro columns, as described above.

Second proteolytic digestion with trypsin Desalted samples were dried completely and dissolved in 50 mM ABC, 1 mM CaCl2 for tryptic digestion. Trypsin was added (~ 1:20 w/w) and incubated overnight at 37 °C.

2nd Dimension Enrichment of Cross-Linked Peptides All 2nd dimension SCX separations were performed using a U3000 HPLC system (Thermo Scientific) and a the smaller 150 x 0.3 mm POLYSULFOETHYL A column (PolyLC INC, Columbia, US, 5 µm particle size, 200 Å pore size). The same buffer system with the optimized gradient as described above was used. Four fractions à 10 min were collected from minute 51 to 91, corresponding to the elution windows of peptides with net charge four and above. Fractions were dried and desalted with self-made microcolumns with Oligo-R3 material as described above. 100% of pBSA, 10% of mBSA and 30% of 8


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hBSA fractions were measured. The enrichment for each sample type (pBSA, mBSA, hBSA) was performed in three individual replicates. SEC-ChaFRADIC of cross-linked peptides We compared our two-dimensional SCX-based ChaFRADIC workflow to an alternative strategy. Here, the workflow was the same as XL-ChaFRADIC, but instead of SCX the first dimension comprised a SEC-based separation with a micro column. The column was equilibrated with 0.1 % TFA and the sample (hBSA) was loaded. By stepwise centrifugation eight fractions were collected (50 g for 15 s, 100 g for 30 s, 200 g for 30 s, 300 g for 30 s, 400 g for 30 s, 500g for 30 s, 600 g for 15 s, 800 g for 1 min). The peptide length distribution obtained from LC-MS showed that peptides with more than 15 amino acids were enriched in fractions two, three, four and five (data not shown). These fractions were pooled for trypsin digestion and 2nd dimension SCX separation.

Proteolytic digestion of control samples (without enrichment) To evaluate the efficiency of XL-ChaFRADIC to enrich cross-linked peptides, we additionally prepared corresponding control samples (pBSA, mBSA, hBSA) which were directly digested with trypsin, without any preceding or subsequent enrichment. Samples were dried in a vacuum centrifuge and dissolved in 4 M GuHCl. Cysteines were reduced with 10 mM DTT for 30 min at 56 °C. Afterwards, free sulfhydryl groups were alkylated using 20 mM IAA for 30 min at RT in the dark. Samples were diluted to a final concentration of 0.2 M GuHCl with 50 mM ABC buffer and 1 mM CaCl2. Trypsin was added in a 1:20 (w/w) ratio and incubated overnight at 37 °C. Digestion was quenched with 1% TFA (final concentration). Digestion efficiency was controlled by monolithic RP separation as described previously16. Samples were desalted as described above with Oligo-R3 microcolumns. 2 pmol of the pBSA control and 300 ng each of the mBSA and hBSA controls were analyzed by LC-MS, in triplicate.

Nano-LC-MS/MS

9



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Samples were analyzed on an Orbitrap Elite or Orbitrap Velos Pro (Thermo Scientific) online coupled to a nano RSLC HPLC (Thermo Scientific). Samples were loaded with 0.1% TFA onto a trap column (Acclaim C18 PepMap100, 100 µm x 2 cm, Thermo Scientific) at a flow rate of 20 µL/min, followed by separation on a RP main column (Acclaim C18 PepMap100, 75 µm x 50 cm or 15 cm, Thermo Scientific) using a binary gradient of solvent A (0.1 % FA) and solvent B (0.1 % FA, 84 % acetonitrile). The gradients increased linearly from 3-50 % B at a flow rate of 250 nL/min in 35 min (SCX fractions) or 3-40% B at a flow rate of 250 nL/min in 60 min (non-enriched samples). Mass spectrometers were operated in data-dependent acquisition mode acquiring full MS survey scans in the orbitrap at R=60,000 using the polysiloxane at 371.101236 m/z as lock mass, followed by collision-induced dissociation (CID) of the 10 most abundant ions with a normalized collision energy of 35. AGC target values and maximum injection times were set to 106 and 50 ms for MS and 104 and 100 ms for MS/MS scans, respectively. A dynamic exclusion of 30 s was used. Only precursor ions with charge states above +2 were selected for fragmentation.

Data analysis with pLink Data analysis was performed with pLink20. Raw-files were converted to mgf format with ProteoWizard msConvertGUI21. Files were searched against a FASTA database containing the BSA sequence. Following parameters were set in the pLink.ini file: sample1.spectra.instrument=CID, fixed modification carbamidomethylation at cysteine, variable modification oxidation at methionine, crosslinker BS3 and BS3-heavy, Filter_peptide_tol_base= 0, filter peptide_tol_lb/_ub= -10/10 ppm. Other parameters were set to default.

Data evaluation For data evaluation, pLink results were imported into Excel. pLink hits with scores > 1e-01 were excluded, as these can be mainly attributed to false positive identifications. Moreover, only PSM with mass deviations < 3 ppm were considered and putative cross-linking PSM, MS1 spectra were checked manually for the presence of an isotopic pattern as expected from the usage of BS3-d0/4. 10


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Finally, we evaluated the backbone-distances of potentially cross-linked lysine residues in the available 3D structure of bovine serum albumin (PDB 4F5S22). Notably, all cross-linking hits involving Lys residues that exceed the maximum distance constraints were excluded based on the aforementioned criteria. Charge state distribution in SCX separation To control the efficiency of charge separation on the SCX column, the potential net charge state distribution at pH 2.7 was regularly evaluated with a tryptic HeLa digestion (one positive charge per free Lys/Arg/His residue and N-terminus). HeLa cells were lysed and SDS was removed as described above. The lysate was dissolved in 4 M GuHCl and carbamidomethlyated with DTT and IAA as described above. Digestion with trypsin (1:20 trypsin : protein) was performed in 50 mM ABC buffer with 1 mM CaCl2 at 37 °C overnight. HeLa peptides were desalted with C18 Spec tips as described above and dried completely. The peptides were reconstituted in SCX buffer A. For the 150 × 1 mm POLYSULFOETHYL A column 100 µg, for the 150 x 0.3 mm POLYSULFOETHYL A column 10 µg of digested HeLa lysate were injected. Throughout the entire gradient 5-min fractions got collected and without any further desalting 1% of each fraction was analyzed by LC-MS. As our experience shows, the charge state distribution can vary between columns and therefore should be checked regularly; gradients and fractionation schemes may require according adaptations. SCX columns also show aging effects over time. Since no synthetic reference cross-linked peptides were available, time windows fraction collection were defined based on the results of the 5 min HeLa results above.

Results and Discussion Efficiency of cross-linking reaction There is no specific information about the efficiency of cross-linking reaction available today. To assess this question, we compared the intensities of peptides involved in cross-links in three non-crosslinked and three cross-linked BSA samples; cross-linking and LC-MS in randomized order were performed as described above. 11



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We selected peptides that only contributed to a single cross-link and which could also be detected in the non-linked sample (table 1). Notably, for the evaluated four peptides the yield of the cross-linking reaction was between 1% and 50%. As expected, the position of the respective peptides within the 3dimensional BSA structure strongly influenced the cross-linking reaction: lysines close to the surface had much higher cross-linking yields than lysines close to the center of BSA. Table 1: To assess the efficiency of cross-linking, we selected three linear peptides that were detected in both sample sets, BSA pure and cross-linked BSA, and compared their intensities to determine the amount of cross-linked peptide. pure BSA (control)

cross-linked BSA

mean crosslocalization in

cross-linked amino

BSA

acids

selected peptide

area XIC (mean)

area XIC (mean)

linking efficiency

center

K187-K280

EccDKPILEK

2.23E+08

2.13E+08

95%

outside

N-terminus-K12

FKDLGEEHFK

1.32E+09

6.85E+08

52%

outside

K350-K474

7.91E+06

4.91E+06

62%

LAKEYEATLEEccA K

Recovery and depletion of peptides in XL-ChaFRADIC An important step in an enrichment procedure such as XL-ChaFRADIC is to evaluate how efficiently matrix components can be depleted, i.e. here non-linked peptides. To do so, we took the results of the hBSA samples, which were measured in triplicate before and after enrichment as described above, and compared

the

intensities

of

non-linked

BSA

peptides.

Raw-files

were

searched

via

ProteomeDiscoverer version 1.3 (Thermo Scientific) using Mascot 2.4 (Matrix Science). Files were searched against the complete Uniprot database (December 2013, 541,954 target sequences) using trypsin as enzyme with a maximum of 2 missed cleavages and with 10 ppm precursor and 0.5 Da fragment mass tolerances. Carbamidomethylation of cysteine (+57.0214 Da) was set as static, oxidation of methionine (+15.9949 Da), mono-links of BS3-d0/4 at lysine (+156.07864 Da) and protein N-termini (+156.0786 Da) were set as dynamic modifications. As the low complexity of the enriched

12


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fractions impairs the resolution of the FDR assessment23 all peptides passing a medium confidence filter (5% FDR, PSMs) were considered for the following analysis. Without enrichment, 48 non-linked BSA peptides were found in at least two out of three replicates. Of those, 31 peptides (64.6 %) could not be identified using the same search settings after enrichment, whereas 17 peptides (35.4 %) were still present. At pH 2.7, 16 of those 17 peptides (96 %) have theoretical net charge states above +2, and thus can be expected in background as cross-linked peptides are collected in the second dimension of SCX at the end of fraction +3 and the beginning of fraction +4. 11 of those 16 peptides (68.2 %) had also high charge states after LysC digestion (> +4) and eluted in the first dimension with the cross-linked peptides and cannot be avoided to be in the final fractions. Importantly, although those peptides could still be identified, their intensities were substantially decreased – for some up to 99.8 %. After enrichment 13 linear peptides were identified in at least two out of three replicates, which could not be identified in the control. Of those 10 should have the same net charge states as cross-linked peptides in both dimensions resulting in co-elution after LysC and trypsin digestion. There are just 3 peptides co-eluting unspecifically, yielding an enrichment specificity of 76.9 %. This data is supported by the theoretical charge state distribution of BSA peptides after LysC and trypsin digestion as depicted in figure 3. Most peptides co-eluting with cross-links in the first dimension of SCX will, after subsequent trypsin digestion, shift to fractions corresponding to substantially lower net charge states during the second dimension and thus will be depleted from the later cross-linked peptides-containing fractions. In contrast the identified cross-linked peptides have theoretical net charge states at pH 2.7 of six and higher, which will be reduced to 4-5 upon digestion with trypsin (figure 4). In contrast, the BSA cross-linked peptides identified in this study have a minimum 1st dimension theoretical net charge of six and mostly retain 4-5 positively charged residues after trypsin digestion (figure 5) such that most linear peptides generated during the second digestion can be separated.

Comparison of trypsin digestion with trypsin-LysC digestion 13



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To investigate the impact the double digestion strategy on the identification of cross-linked peptides, we used a pBSA control sample which was either digested with trypsin or with LysC followed by trypsin, each three times, and analyzed the samples for the occurrence of cross-links, however without enrichment (table 2). Two cross-links (NT1-K261 and K187-K280) could only be found with the double digestion procedure.

Table 2: Identified cross-link peptides in pBSA control (without enrichment) after trypsin and LysC-trypsin digestion. REP1-3 = replicate 1-3, numbers represent pLink scores. “x” = identified in at least two out of three replicates. “xx” = only identified with LysC-trypsin identification.

pBSA trypsin REP1

REP2

pBSA LysC-trypsin

AA1

AA2

distance (Å)

REP3

1

12

19.5

1

261

25.4

116

431

21.3

1.09E-03

2.83E-03

2.18E-04

180

187

11.0

1.34E-05

6.15E-06

187

439

18.2

1.47E-05

187

221

20.3

187

431

13.7

187

280

20.0

204

350

16.4

3.78E-02

221

439

20.5

1.21E-02

431

439

13.5

8.75E-06

2.26E-06

524

544

14.3

2.78E-04

1.05E-04

REP1

1.27E-04

REP2

REP3

5.77E-04 1.48E-04

2.88E-04

1.03E-03

xx

x

6.59E-04

3.57E-05

1.00E-03

x

3.50E-06

x

3.24E-04

3.98E-05

1.67E-08

3.13E-04

x

9.12E-06

3.63E-04

8.37E-06

x

1.32E-04

1.99E-04

5.91E-05

x

4.83E-05

7.85E-05

6.31E-06

x

3.40E-04

5.62E-04

7.31E-04

x

3.04E-04

8.07E-03

3.61E-03

x

4.47E-04

4.93E-05

2.99E-03

xx

3.96E-06

x

1.57E-04

x

3.89E-05

x

2.89E-04

6.39E-04

8.30E-05

x

7.79E-06

x

1.68E-06

3.89E-04

2.35E-03

x

x 8

5.96E-04 10

Enrichment of BSA crosslinks – pBSA sample Table 3: Cross-linked peptides identified in pBSA without and with XL-ChaFRADIC enrichment. Numbers represent p-Link e-values. AA: amino acid; REP=replicate. High confidence hits found in at least two out of three replicates are labeled with “x”. Cross-links only identified after XL-ChaFRADIC enrichment are labeled with “xx”. Hits in brackets may be caused

14


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by LysC-trypsin digestion rather than enrichment. Even with the pure BSA, ChaFRADIC led to the identification of additional cross-links compared to a tryptic digestion of cross-linked BSA without ChaFRADIC enrichment. direct analysis of pBSA (tryptic) AA1

AA2

distance (Å)

REP1

REP2

1

12

19.5

1.80E-04

1.34E-03

12

131

114

pBSA with XL-ChaFRADIC

REP3

REP1

REP2

REP3

3.62E-06

4.11E-06

1.78E-05

20.4

8.34E-04

4.06E-04

431

19.9

1.81E-04

1.36E-03

2.99E-02

xx

116

431

21.3

3.10E-04

1.15E-03

4.29E-05

x

2.86E-04

9.83E-04

2.11E-05

x

180

187

11.0

6.62E-05

3.67E-05

2.81E-04

x

1.84E-04

1.23E-03

x

187

221

20.3

1.75E-04

7.09E-04

1.52E-05

x

8.13E-06

4.15E-05

2.94E-06

x

187

280

20.0

5.08E-06

4.16E-04

6.99E-06

(xx)

187

431

13.7

4.05E-04

3.57E-04

5.28E-04

x

3.70E-04

7.27E-05

8.00E-04

x

187

439

18.2

8.18E-08

7.97E-07

2.76E-06

x

3.60E-06

1.38E-05

9.48E-07

x

204

350

16.4

6.97E-03

5.43E-03

211

239

9.0

8.74E-04

7.41E-03

221

439

20.5

2.74E-04

2.26E-04

2.54E-04

x

1.89E-03

7.29E-03

350

474

18.0

1.80E-05

4.98E-08

5.82E-07

x

431

439

13.5

8.28E-05

3.94E-07

1.05E-05

x

4.50E-05

524

544

14.3

2.62E-06

2.43E-04

7.37E-04

x

7.45E-05

x

x xx

x xx 1.58E-03

x

5.38E-05

11

6.85E-05

3.57E-04

x

2.28E-02

x 13

For pBSA we could identify four additional cross-linked peptides after XL-ChaFRADIC as compared to the strictly trypsin-digested controls (table 3). Indeed, as pure BSA is a sample of low complexity, we did not expect a high increase in the number of cross-linked peptides. However, not only could we identify three additional cross-links, but we could also increase the number of well-scoring PSMs for two cross-linked peptides, which improves the reliability of the identifications. The identified crosslinks are represented in a 3D structure of BSA in figure 2. To show the advantage of our two-step enrichment procedure, we compared cross-linked peptides identified after a one-step SCX separation of trypsin-digested pBSA with the results of the ChaFRADIC enrichment. 15



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16


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4: Cross-linked BSA peptides identified after a single SCX separation and after XL-ChaFRADIC. Numbers represent p-Link e-values. AA: amino acid; REP=replicate. High confidence hits found in at least two out of three replicates are labeled with “x”. Cross-links only identified after any enrichment method are labeled with “xx”. Notably, K187-K280 was also found after sole trypsin digestion 1D-SCX separation such that the identification of this cross-link does not merely depend on the two-step digestion procedure. 1D SCX separation

1D-SCX

ChaFRADIC

3 replicates

3 replicates

x

x

AA1

AA2

distance (Å)

REP1

REP2

REP3

1

12

19.5

9.17E-06

2.83E-06

3.22E-06

12

131

20.4

xx

114

431

19.9

xx

116

431

21.3

2.00E-05

2.44E-05

6.17E-04

x

x

x

180

187

11.0

2.27E-04

2.33E-05

2.14E-04

x

x

x

187

221

20.3

1.62E-04

2.59E-06

6.56E-05

x

x

x

187

280

20.0

5.90E-06

2.63E-04

xx

187

431

13.7

3.51E-04

1.71E-05

2.28E-04

x

x

x

187

439

18.2

2.31E-05

3.07E-08

1.08E-06

x

x

x

204

350

16.4

1.27E-04

1.30E-04

1.94E-06

x

x

211

239

9.0

221

439

20.5

1.91E-07

6.46E-06

1.02E-04

x

x

350

474

18.0

2.76E-05

1.94E-07

1.28E-03

x

x

350

377

13.1

3.62E-07

6.14E-05

xx

431

439

13.5

2.79E-06

5.30E-04

x

524

544

14.3

x

xx

xx

3.18E-05

12

x

x

x

x

x

11

13

Using pure BSA the 1D SCX separation led to the identification of two additional cross-links compared to controls, whereas ChaFRADIC yielded 4 additional cross-linked peptides. Although we assumed before, that the cross-link K187-K280 was just identified because of the double digestion strategy, the 1D-SCX separation showed that this identification is rather due to enrichment.

17



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Page 18 of 33

Enrichment of BSA crosslinks from protein mixtures – mBSA sample Table 5: Cross-linked peptides identified in mBSA without and with XL-ChaFRADIC enrichment. Numbers represent pLink e-values. AA: amino acid; REP=replicate. High confidence hits found in at least two out of three replicates are labeled with “x”. Cross-links only identified after XL-ChaFRADIC enrichment are labeled with “xx”. Hits in brackets may be caused rather by double digestion than enrichment.. For BSA with a protein background, ChaFRADIC led to the identification of additional cross-links compared to a tryptic digestion of cross-linked BSA without ChaFRADIC enrichment.

direct analysis of mBSA (tryptic) AA1

AA2

distance (Å)

REP1

REP2

REP3

1

12

19.5

8.00E-06

2.15E-05

3.45E-05

1

261

114

mBSA with XL-ChaFRADIC REP1

REP2

REP3

3.21E-05

3.81E-06

2.02E-05

x

25.4

1.61E-04

2.41E-05

3.01E-04

(xx)

431

19.9

1.14E-04

8.33E-04

3.36E-04

xx

116

431

21.3

4.67E-04

3.94-04

1.98E-04

x

5.52E-04

5.31E-05

187

221

20.3

5.40E-04

1.93E-04

2.27E-04

x

2.83E-05

6.22E-05

7.90E-04

x

187

431

13.7

4.31E-04

3.88E-06

5.67E-05

x

3.21E-03

8.66E-06

1.43E-04

x

187

439

18.2

6.67E-05

1.64E-04

3.85E-06

x

1.02E-04

5.14E-04

x

211

350

13.5

3.34E-06

1.05E-05

3.60E-07

x

221

239

19.3

8.32E-03

xx

221

439

20.5

8.39E-05

1.46E-04

3.42E-04

431

439

13.5

1.44E-06

1.80E-04

1.66E-05

524

544

14.3

1.61E-06

x

1.60E-04

1.13E-04

x

3.69E-03

1.36E-04

x

4.17E-04

7.63E-05

x 9

x

x 4.52E-05

x

5.24E-05 10

As summarized in table 5, for mBSA we could identify two additional cross-linked peptides compared to the control sample. Although XL-ChaFRADIC comprises several steps, a reproducible enrichment is possible, as depicted in figure 5 which represents replicate base peak chromatograms of the four fractions collected in the 2nd dimension of SCX.

18


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Enrichment of BSA crosslinks with complex HeLa background – hBSA sample Table 6: Cross-linked peptides identified in hBSA without and with XL-ChaFRADIC enrichment. Numbers represent p-Link e-values. AA: amino acid; REP=replicate. High confidence hits found in at least two out of three replicates are labeled with “x”. Cross-links only identified after XL-ChaFRADIC enrichment are labeled with “xx”. For BSA with a protein background, ChaFRADIC led to the identification of additional cross-links compared to a tryptic digestion of cross-linked BSA without ChaFRADIC enrichment.

direct analysis of hBSA (tryptic) REP1

REP2

hBSA with XL-ChaFRADIC

AA1

AA2

distance (Å)

REP3

REP1

REP2

REP3

1

12

19.5

8.45E-06

2.26E-06

5.61E-07

xx

1

239

22.7

2.30E-04

1.00E-05

xx

12

131

20.4

4.09E-05

1.28E-04

1.84E-05

xx

12

280

21.0

3.96E-04

6.39E-06

116

431

21.3

4.50E-05

1.70E-04

5.57E-04

x

1.20E-04

2.07E-05

2.87E-04

x

180

187

11.0

7.27E-04

2.33E-04

7.20E-06

x

2.26E-05

5.22E-05

1.78E-05

x

187

221

20.3

7.95E-04

6.69E-04

8.16E-05

x

5.56E-05

1.26E-04

5.36E-05

x

187

280

20.0

4.43E-06

4.09E-04

3.65E-06

xx

187

431

13.7

2.71E-04

xx

187

439

18.2

211

350

13.5

2.88E-06

221

239

19.3

1.94E-03

221

439

20.5

1.31E-02

7.72E-05

350

474

18.0

2.58E-04

1.79E-05

8.98E-05

x

431

439

13.5

5.80E-06

1.26E-05

2.90E-05

x

524

544

14.3

4.61E-05

3.77E-05 8.32E-05

9.04E-06

5.02E-05

xx

6.38E-07

x

6.06E-07

6.48E-06

8.67E-06

x

5.33E-05

x

5.68E-05

1.85E-05

3.61E-05

x

x

3.60E-04

3.68E-04

1.96E-04

x

1.86E-05

4.64E-05

4.55E-05

x

3.83E-05 8

13

For hBSA we identified six cross-links which could not be found in the control sample without XLChaFRADIC enrichment (table 6). Besides this also the number of PSMs for four other cross-linked peptides was increased, which makes the identifications more reliable. Three cross-linked peptides (K1-K12, K187-K431 and K524-K544) showed low reproducibility of identification in hBSA without 19



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Page 20 of 33

enrichment. For those cross-links, the XL-ChaFRADIC approach additionally improved the significance of the identification.

Comparison with SEC-SCX As shown in table 7, the alternative SEC-SCX approach was less effective in the enrichment of crosslinked peptides. The results are comparable to the XL-ChaFRADIC workflow, but with XLChaFRADIC in total more cross-links were identified. However, in general the combination of our two-step digestion approach with (i) two-dimensional SEC or (ii) HPLC-based SEC followed by SCX might be promising alternatives, as the number of identified cross-links after offline SEC and HPLCbased SCX is not dramatically lower than after XL-ChaFRADIC. SEC has the same advantage of being universally applicable to every kind of cross-linking reagent as SCX separation. Especially the usage of SEC-HPLC instead of micro columns should be considered for future work11. Table 7: Identified cross-linked peptides in hBSA with SEC-SCX enrichment and XL-ChaFRADIC enrichment. Numbers represent p-Link e-values. AA: amino acid; REP=replicate. hBSA without

hBSA with

ChaFRADIC

ChaFRADIC

score (mean)

score (mean)

REP1

REP2

REP3

3.22E-06

1.20E-05

3.44E-05

1.45E-03

7.65E-05

3.86E-04

1.44E-04

hBSA SEC-SCX

AA1

AA2

distance (Å)

1

12

19.5

3.76E-06

1

239

22.7

1.20E-04

12

131

20.4

6.24E-05

12

280

21

2.01E-04

114

431

19.9

116

431

21.3

2.57E-04

1.43E-04

180

187

11

3.22E-04

3.09E-05

187

221

20.3

5.15E-04

7.84E-05

1.62E-04

3.05E-05

187

280

20

1.39E-04

2.69E-03

4.82E-05

187

431

13.7

1.61E-04

3.26E-04

2.86E-04

2.23E-04

187

439

18.2

3.10E-05

5.25E-06

1.95E-04

2.99E-05

1.37E-04

221

239

19.3

9.97E-04

3.71E-05

2.78E-04

2.54E-04

1.82E-04

1.57E-05 2.95E-04

20


1.43E-04

Page 21 of 33

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221

439

20.5

6.59E-03

350

474

18

1.22E-04

431

439

13.5

1.58E-05

524

544

14.3

3.08E-04

5.91E-06

8.25E-05

4.95E-06

3.68E-05

1.21E-04

1.01E-05

1.32E-06 3.71E-05

8

13

10

10

10

Conclusion In summary, our novel XL-ChaFRADIC workflow for the enrichment of cross-linked peptides allows an effective enrichment of cross-linked peptides. We showed the application of the workflow for three samples of different complexity and for each sample type yielded additional identifications for crosslinked peptides. Additionally, we could increase the pLink identification scores for many cross-links, which correlates with a higher spectrum quality and consequently a more confident identification of cross-linked peptides. With XL-ChaFRADIC we also significantly decreased the number of nonlinked and mono-link peptides, which usually hamper the identification of cross-links. We envision that thus ChaFRADIC may help to identify system-wide cross linked in whole cell cross-linking studies, as here (i) sufficient starting material is available to conduct the workflow on a large 1st dimension SCX column and (ii) an effective depletion of the large bulk of non-linked and monolinked peptides will be essential (>50% reduction in the number of identified mono-linked peptides achieved, data not shown). Notably, our approach is independent of the used cross-linker and no specialized cross-linking reagents are needed for the enrichment. The comparison with SEC micro columns indicated the potential of this combination as an alternative and simple approach, although XL-ChaFRADIC provided superior results. Nevertheless, a twodimensional SEC approach would allow the same universal application and future work could focus on the combination of both methods for large scale cross-linking studies. A possible improvement could be the use of a SEC HPLC system instead of micro columns.

21



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SUPPORTING INFORMATION: The following files are available free of charge at ACS website http://pubs.acs.org: Supporting Information.pdf. Supplementary Table 1: Theoretical charge state distribution of linear BSA peptides generated with LysC.

(page S2)

Supplementary Table 2: Cross-linked peptide with calculated charge states after LysC digestion and following trypsin digestion. (page S5) Supplementary Table 3: Identified linear peptides of hBSA with their expected net charge states at pH 2.7 in control and/or ChaFRADIC samples. (page S7) Supplementary Table 4: Peptide sequences of cross-links found in pBSA, mBSA and hBSA before and after enrichment.

(page S11)

22


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Figures Figure 1. Schematic workflow of the XL-ChaFRADIC approach.

23



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Figure 2. 3D structure of BSA (PDB 4F5S) with found cross-links (yellow). Additional cross-links identified with XL-ChaFRADIC are blue. Lysines are red.

24


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Figure 3. Theoretical charge state distribution of linear BSA peptides (determined upon number of positively amino acid residues) generated with LysC (charge states +4 and higher) in the 1st dimension (bottom) and the resulting tryptic peptides (top) in the 2nd dimension. Tryptic peptides are classified by the theoretical charge states of LysC peptides they were derived from. In silico digested with LysC or trypsin, minimum of five amino acids, no modifications, one allowed missed cleavage (Suppl. Table 1).

25



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Figure 4.

Theoretical charge state distribution of cross-linked BSA peptides (determined upon

number of positively amino acid residues) generated with LysC and the resulting tryptic cross-linked (XL, shades of blue) and cleaved off, linear peptides (red) in the 2nd dimension. Resulting tryptic peptides are classified by the theoretical charge states of LysC peptides they were derived from. In silico digested with LysC or trypsin, minimum of five amino acids, no modifications, one allowed missed cleavage (Suppl. Table 2).

26


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Figure 5. Base peak chromatograms of the four fractions (fraction 3—6) of the 2nd dimension of SCX separation, which were measured for data analysis with pLink for mBSA samples. 3 replicates are shown in black, red and green.

27



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ACKNOWLEDGMENT Financial support by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin and the Bundesministerium für Bildung und Forschung is gratefully acknowledged.

ABBREVIATIONS ABC

ammonium bicarbonate

BCA

bicinchoninic acid

BS3-d0/4

bis(sulfosuccinimidyl) 2,2,7,7-suberate-d0/4

BSA

bovine serum albumin

ChaFRADIC

charge based fractional diagonal chromatography

DMSO

dimethylsulfoxid

DTT

dithiothreitol

EDTA

ethylenediaminetetraacetic acid

GuHCl

guanidine hydrochloride

hBSA

cross-linked BSA in HeLa lysate

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IAA

iodoacetamide

LC-MS

liquid chromatography mass spectrometry

mBSA

cross-linked BSA in protein mixture

pBSA

cross-linked BSA

SCX

strong cation exchange chromatography

SDS

sodium dodecyl sulfate

SEC

size exclusion chromatography

Tris

tris-(hydroxymethyl)-aminomethane

XL-MS

cross-linking mass spectrometry 28


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Table of contents graphic Figure 1. Schematic workflow of the XL-ChaFRADIC approach. Figure 2. 3D structure of BSA (PDB 4F5S) with found cross-links (yellow). Additional cross-links identified with XL-ChaFRADIC are blue. Lysines are red. Figure 3. Theoretical charge state distribution of linear BSA peptides (upon number of positively amino acid residues) generated with LysC with theoretical charge states of +4 and higher in the 1st dimension (bottom) and the resulting tryptic peptides (top) in the 2nd dimension. Tryptic peptides are divided by the theoretical charge states of LysC peptides they were derived from. In silico digested with LysC or trypsin with minimum peptide length of five amino acids. No modifications and one missed cleavage site are respected. Figure 4.

Theoretical charge state distribution of cross-linked BSA peptides (upon number of

positively amino acid residues) generated with LysC (middle bar diagram) and the resulting tryptic cross-linked (XL, shades of blue) and cleaved off, linear peptides (red) in the 2nd dimension. Resulting tryptic peptides are divided by the theoretical charge states of LysC peptides they were derived from. In silico digested with LysC with minimum peptide length of five amino acids. No modifications and one missed cleavage site are respected. Figure 5. Base peak chromatograms of the four fractions (fraction 3—6) of the 2nd dimension of SCX separation, which were measured for data analysis with pLink for mBSA samples. 3 replicates are shown in black, red and green.

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Enrichment of Cross-Linked Peptides Using Charge-Based Fractional

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