ISSN: 2155-9872

Journal of Analytical & Bioanalytical Techniques
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ESI-MS and Stavrox 3.6.0.1 Investigations of Crosslinking by an Aryl-Azido-NHS-Heterobifunctional Crosslinker

Sujeet Thakur K and Eswaran SV*
Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana, India
*Corresponding Author: Eswaran SV, Regional Centre for Biotechnology (Established by DBT, Government of India under the auspices of UNESCO), NCR Biotech Science Cluster, 3rd Milestone, Faridabad- Gurgaon Expressway, Faridabad, Haryana-121 001, India, Tel: 8860077657, Email: samba.eswaran@rcb.res.in

Received: 12-Mar-2018 / Accepted Date: 23-Mar-2018 / Published Date: 26-Mar-2018 DOI: 10.4172/2155-9872.1000402

Abstract

Chemical cross-linking-mass spectrometry (CX-MS) combined with bioinformatics tools is increasingly being used to analyze large-scale protein–protein interactions. It has gained importance in studies in proteomics, lipidomics, in systems and structural biology. Recently it has gained importance in preparation of homogeneous antibody-drug conjugates, which has been described as “a pinnacle of such targeting efforts.” What makes these approaches exciting is that using the “Click” and Bertozzi protocols in vivo studies can be carried out successfully. Using CX-MS combined with cryo-EM, structures of protein complexes can now be probed at almost molecular resolution (upto 3 Å). Chemical crosslinking is useful in materials science, as well. Major advances in both mass spectrometric techniques and bioinformatics tools today allow one to identify cross-linked peptides with highconfidence and with more user-friendly approaches. Crucial to this is the ability to capture intermolecular crosslinking reliably.

The use of a new small NHS-aryl azido heterobifunctional cross-linker based is described here, which picks intermolecular crosslinking better. Thus, Lysozyme has been crosslinked successfully as established by the ‘dimeric’ band observed in SDS-PAGE. its tryptic digestion, ‘zip tip’ enrichment, ESI-MS, MS/MS and the data generated analyzed using StavroX 3.6.0.1, a bioinformatics software, especially suited for determining intermolecular crosslinking.

Keywords: Chemical crosslinking; SDS-PAGE; ESI-MS; MS/MS; StavroX 3.6.0.1

Introduction

Chemical-crosslinking-mass spectrometry-bioinformatics has become an important technique for studying large scale protein-protein interactions, especially for capturing transient interactions [1-18]. This has become possible by the availability of a wide variety of crosslinkers, viz. both homo-and hetero-bifunctional crosslinkers. The former has identical displaceable groups on the two ends, e.g., the amine displaceable N-hydroxysuccinimide (NHS) group. One of more popular reagent of this type is BS2G (bis[sulfosuccinimydyl]glutarate), which has been used extensively.

For enhancing intermolecular crosslinking, more efficient crosslinkers are required. This limitation is overcome by using heterobifunctional crosslinkers, where two different groups are present on the two ends. One of these groups being thermally reactive (e.g., the NHS group) and the other one being photoreactive (e.g., the azide group), which also provides greater flexibility in experimental protocols. Small crosslinkers are known to be more effective for mapping interfaces, while the larger crosslinkers are more useful for identifying binding sites. X-ray diffraction and NMR continue to be the gold standards, but both have their own limitations. The former requires a single crystal, while the latter requires solubility in specific solvents and also demands larger amounts of the sample. Thus, both these techniques are not yet suitable for dynamic studies.

Despite the availability of a range of crosslinkers (including cleavable and isotope labeled crosslinkers) many laboratories continue to use conventional crosslinkers like formaldehyde, glutaraldehyde as crosslinkers, even though these lead to undesirable and extensive crosslinking. Even “zero crosslinkers” like DCC are still in use. This arises mainly due to the lack of awareness amongst many biochemists and others of the immense potential of this new technique of ‘chemical crosslinking mass spectrometry-bioinformatics’ particularly for studying dynamic interactions in living cells. It has been proposed that structures and functions of large protein complexes at the molecular or atomic level in dynamic situations can be studied by combining cryo-electron microscopy (cryo-EM) with crosslinking- mass spectrometry (CX-MS) [19].

Traditional crosslinking strategies generate an enormous amount of mass spectrometry data, which is extremely difficult to analyze with routine software tools. The situation has often been compared to “finding a needle in a haystack”. Tremendous advancement in mass spectrometry (MALDI-MS, MS/MS, ESI-MS) have provided much impetus in improving the quality of crosslinking studies. The use of different algorithms in each laboratory has led to better bioinformatics tools such as GPMAW, MSX3D, ProteinPilot, XQuest, ExPASy, StavroX, xTract. These have contributed greatly to making this technique a valuable tool for the identification of protein-protein interactions in dynamic systems.

The protocol (Figure 1) commonly used for heterobifunctional crosslinkers involves, incubating the first protein with the crosslinker at room temperature and then in the second step the incubated sample is subjected to photolysis. In the second step, which could be done using either 254 nm or 366 nm UV lamp, a second and a different protein could be introduced. Shorter wavelengths could potentially damage proteins, while longer wavelength exposures are safer.

analytical-bioanalytical-techniques-bioinformatics

Figure 1: Schematic representation of the chemical cross-linking-mass spectrometry-bioinformatics methodology.

Much optimization of the duration of incubation, ratio of protein to the crosslinker ratio; exposure time, etc. are required before experimental success can be ensured. The sample at this stage is then subjected to SDS-PAGE. To establish intermolecular crosslinking, the ‘dimeric’ band is excised, trypsin digested and subjected to mass spectrometric analysis. The huge amount of data thus generated is then analyzed by suitable bioinformatics tools. As most of the protein (s) is not crosslinked, unlabeled peptide fragments usually dominate. Unlike the latter, labeled peptide fragments carry a positive charge (+1, +2, +3…) and can be thus enriched by strong cation exchange(SCX) (‘zip tip’) tips to separate them from the un-crosslinked fragments, which usually do not carry a charge. This has been referred to as the “double needle in a haystack” problem.

We employed the above protocol with the twin aims of confirming whether our new small heterobifunctional crosslinker does indeed bring about intermolecular crosslinking successfully and whether the same could be established using modern mass spectrometric methods (MALDI-MS, MS/ MS; ESI-MS) combined with StavroX 3.6.0.1. a bioinformatics software, especially suited for identifying intermolecular crosslinks reliably.

Our work arose out of Hagan Bayley’s [20] correct prediction based on the work of Banks et al. [21] on perfluorophenylazides, that reagents based on the latter could serve as efficient photo-affinity labelling agents, as such reagents involve ‘long-lived ’transients, which leads, in turn, to enhanced intermolecular reaction rates. This was confirmed by Platz et al. [22] who also showed that in this case, the singlet-triplet nitrene energy gap increases. The involvement of a “slippery potential energy surface” has also been demonstrated [23]. The concepts described above have found applications in diverse areas [24].

We have also published a series of papers in this area. Our reactions, however, do not involve any fluorination, which is a demanding step and yet we obtain comparable results. Further, in our cases, the initially formed singlet nitrene does not flip to the triplet state and instead forms the corresponding carbene (Nitrenecarbene conversion; Crow-Wentrup pathway) [25], which has also been substantiated by computational studies, yet our results parallel those based on perflourinated aryl azides. We first observed an unusual reaction involving a concomitant ring expansion and ring extrusion during thermolysis of ‘Dimethyl-Azido-m-hemipinate’ [26]. This was followed by establishing “nitrene insertion into an adjacent orthomethoxy group followed by the addition of the amine intermediate on to the corresponding heterocumulene intermediate” along with the isolation of carbene based products [27]. The life span of one such ‘long lived’ transient, in our case, has been shown to be 700 picoseconds [28]. Reaction of the somewhat more rigid azido-m-meconine led to intramolecular cyclisation via reaction with the adjacent o-methoxy group [29,30]. The reaction of the para analog, viz. ‘Dimethyl-azidosuccinylosuccinate’ led to similar results [31]. Recently we have published on crosslinking studies using a new heterobifunctional crosslinker based on an “introverted” carboxylic acid [32]. The present work is thus a part of our continued investigations in this field.

Results and Discussion

The new aryl azido-N-hydroxysuccinimide heterobifunctional crosslinker (I) was synthesized as shown in scheme I (SI-I). Thus, ‘dimethyl azido-m-hemipinate’ was subjected to selective alkaline hydrolysis, which was followed by reaction with N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) to yield (1), m.p. 165°C. (I) contains a photo-reactive aryl azido (-N3) group and an amine reactive N-hydroxysuccinimide (NHS) moiety, facilitating the two step protocol, viz. an initial incubation step followed by the photolysis step or vice-versa. The structure of (I) was established by detailed spectroscopic studies including, FT-IR, UV, MALDI-MS and MS/MS studies. NMR studies on (I) will be published separately.

Thus, FT-IR spectrum of (I) (in KBr) showed peaks at 3326, 2928, 2850, 2124, 1767, 1742, 1626, 1588, 1498, 1419,1351,1293, 1201, 1127, 1031, 968, 938, 913, 867, 641 cm-1. UV spectrum of (I) (λ max) in MeCN) showed 198 (2.17), 244(2.44), 2.87(0.49), 317(0.22) nm. MALDI-MS spectrum of the new cross-linker (I) (Figure 2) showed the [M+ H+] ion at m/z 379.0935 as the base peak yielding the molecular formula C15H14N4O8. In addition, other major peaks were observed at m/z 306.2756, 239.1829 and 225.1643. The MS/MS spectrum of the m/z 379 peak is given in SI-II.

analytical-bioanalytical-techniques-MALDI

Figure 2: MALDI-MS of (I).

The new heterobifunctional crosslinker (I) was incubated with Lysozyme. This was followed by photolysis at 366 nm (using a 6W TLC visualization UV lamp) and SDS-PAGE (SI-III) with standard protocol (SI-IV,) gave a ‘dimeric’ band at 28 kDa. This confirmed successful intermolecular cross-linking. The ‘dimeric’ band was excised, trypsin digested (SI-V) and enrichment by using zip tip C-18 with standard protocol (SI-VI). In early work on this project, we had no access ‘in house’ mass spectrometric facilities and we received only MALDIMS data with no MS/MS data. Even the bioinformatics software had limitations. In the current centre we had access to all modern MS facilities under one roof. Even here, too, we initially carried out only MALDI-MS (SI-VII and SI- VIII) investigations. We had to switch to ESI-MS, MS/ MS as ESI-MS data ((SI-IX) which works better for the bioinformatics software, StavroX 3.6.0.1, (used by us in the current study) works on ESI-MS data alone. The ESI-MS chromatogram for the crosslinked Lysozyme is shown in (SI-X). The ESI-MS data thus obtained was stored in the form of .mgf file and then fed into the StavroX 3.6.0.1 software (SI-XI).

StavroX 3.6.0.1 software

For analysis, the original FASTA sequence of Lysozyme along with the ESI-MS data as .mgf file was uploaded into StavroX 3. 6. 0. 1 software to identify the intermolecularly cross-linked peptides. StavroX 3.6.0.1 is a recent version of this software and it enables quick and efficient identification of the intermolecularly cross-linked peptides. This software calculates the theoretical cross-links and estimates them to the precursors of the ESI-MS data stored in the form of .mgf file [33]. This further leads to the identification of the hits and scores which are then tabulated. The software provides options to select the desired crosslinker along with the scope to add new cross-linkers. The software itself calculated the actual mass of our new crosslinker as 236.2803 amu, based on the loss of one N-hydroxysuccinimide (NHS) unit and one molecule of N2 (loss of nitrogen from the azide), which are lost during the incubation and the photolysis steps, respectively. No changes were made in the amino acid sequence section. An unspecific digest option was selected along with minimum peptide length as 1 and maximum peptide length as 10. The precursor precision was selected to be 250.0 ppm, fragment ion precision as 1.0 Da with the lower mass limit as 200.0 Da and upper mass limit as 6000.0 Da. The S/N ratio was selected to be 2. Only ‘b’ and ‘y’ ions were selected with the score cut-off of -1 and pre-score intensity greater than 10%.

After the data was uploaded into the StavroX 3.6.0.1 software along with the FASTA sequence of Lysozyme, appropriate settings were selected as stated above and the software was run to allow the data to be processed. As a result, 123 spectral peaks were compared to 1024687 theoretical candidates out of which, 3791 possible cross-links were identified within 1 minute and 17 seconds of the run. Out of these, the top nine crosslinked peptide fragments with their m/ z values and scores for intermolecular crosslinking are shown in Table 1. Out of all the possible cross-linked candidates, the highest score obtained was 98 (for the m/z 1383.523 fragment).

NR SCORE M/Z Z M+H+ Calc. Dev Peptide (1) Protein
(1)
from to Peptide
(2)
Protein
(2)
from to Site
(1)
Site
(1)
rank scan RT
1 98 461.846 +3 1383.523 1383.725 -146 [KIVS] >5K70:A 97 101 [AWRNR] >5K70:A 110 115 S4 R5 1 Locus:1.1 683
2 97 460.264 +3 1378.778 1378.643 97.61 [KVF] >5K70:A 0 4 [CKGTDVQ] >5K70:A 115 122 K1 C1 1 Locus:1.1 1250
3 95 461.948 +3 1382.83 1383.83 75.79 [KIVS] >5K70:A 97 101 [AWRNR] >5K70:A 110 115 S4 R5 1 Locus:1.1 919
4 95 606.885 +2 1212.762 1212.636 104.6 [KK] >5K70:A 96 98 [AWRNR] >5K70:A 110 115 K2 A1 1 Locus:1.1 1129
5 90 771.053 +3 2311.145 2311.109 15.67 [TDVQAWIR] >5K70:A 118 126 [RHGLDNYRG >5K70:A 14 23 T1 G3 1 Locus:1.1 488
6 88 478.792 +4 1912.146 1911.893  132 [QATNR] >5K70:A 41 46 [RHGLDNYRG] >5K70:A 14 23 T3 H2 1 Locus:1.1 1055
7 86 403.862 +5 2015.28 2014.957 160 [NWVCAAK] >5K70:A 27 34 [TDVQAWIR] >5K70:A 118 126 K7 I7 1 Locus:1.1 623
8 85 714.923 +2 1428.84 1428.714 88.08 [AAMKR] >5K70:A 10 15 [QINSR] >5K70:A 57 62 K4 I2 1 Locus:1.1 548
9 85 403.862 +5 2015.28 2014.999 139 [ILQINSR] >5K70:A 55 62 [KIVSDGNGm] >5K70:A 97 106 R7 K1 2 Locus:1.1 623
10 84 48.792 +4 1912.146 1911.824 168.3 [FESNF] >5K70:A 34 39 [MNAWVAWR] >5K70:A 105 113 S3 W4 2 Locus:1.1 1055

Table 1: Major peaks of the crosslinked peptide fragments.

After the analysis was done, a window opened on top, showing a bar chart, where the number of candidates identified in a certain score range (number of hits) to the score hit is plotted. The Decoy analysis figure helped to estimate the quality of the score in our experiment. The blue bars represent the number of candidates from our data set and the red bars represent the number of false positive candidates from a decoy data set, which is obtained from the inverted sequence of the FASTA file (SI-XII). More enriched real data set candidates in the score region indicates towards better crosslinking. The decoy analysis data for fragment at m/z 1404.900 is shown in Figure 3.

analytical-bioanalytical-techniques-decoy

Figure 3: Screen shot of the decoy analysis for m/z 1404.900.

Out of the candidates with high scores, the detailed spectrum for the peak value m/z 1404. 900, the peptide fragments involved in the process of intermolecular cross-linking are shown in Figure 4. The spectrum panel shows the MS2 spectrum for the identified peaks. This one example with its annotation is shown here as a representative. In the deviation diagram (printed below the spectrum panel, deviation of the identified signals is plotted against the m/z values) less deviation in the annotation, points towards better results in the crosslinking experiment. Similar detailed spectra of the other intermolecularly crosslinked fragments have also been obtained via StavroX 3. 6. 0.1, from our experimental data, but these have not been exhibited here (Tables 2 and 3).

analytical-bioanalytical-techniques-intermolecularly

Figure 4: Detailed spectrum of the intermolecularly cross-linked candidate, m/z 1404.900 fragment, with the score of 71, along with its annotation. Modified fragment ions for m/z 1404.900 obtained from the StavroX 3.6.0.1 analysis are shown in Table 2.

b b-H2O b-NH3 AA y y-H2O y-NH3
Peptide: ɑ
Charge: +1
742.39 724.379 725.363 L      
813.427 795.416 796.4 A 663.361 645.35 646.334
884.464 866.453 867.437 A 592.324 574.313 575.297
955.501 937.49 938.474 A 521.285 503.276 504.26
1102.536 1084.526 1085.51 M 450.249 432.239 433.223
1230.631 1212.621 1213.605 K 303.214 285.203 286.187
      R 175.119 157.108 158.092
Charge: +2
371.698 362.693 363.185 L      
407.217 398.212 398.704 A 332.184 323.179 323.671
442.736 433.73 434.222 A 296.665 287.660 288.152
478.254 469.249 469.741 A 261.147 252.142 252.634
551.772 542.767 543.259 M 225.628 216.623 217.115
615.819 606.814 607.306 K 152.111 143.105 143.597
      R 88.063 79.058 79.55
Charge: +3
248.135 242.131 242.459 L      
271.814 265.81 266.138 A 221.792 215.788 216.116
295.493 289.489 289.817 A 198.113 192.109 192.437
319.172 313.168 313.496 A 174.434 168.43 168.758
368.184 362.18 362.508 M 150.755 144.751 145.079
410.882 404.878 405.206 K 101.743 95.739 96.067
      R 59.045 53.041 53.369
Charge: +4
186.353 181.85 182.096 L      
204.112 199.609 199.855 A 166.596 162.093 162.339
221.871 217.369 217.615 A 148.836 144.334 144.580
239.631 235.128 235.374 A 131.077 126.574 126.82
276.39 271.887 272.133 M 113.318 108.815 109.061
308.413 303.911 304.157 K 76.559 72.056 72.302
      R 44.535 40.033 40.279
Peptide: β
Charge: +1
1140.596 1122.585 1123.569 K      
1239.664 1221.653 1222.637 V 265.155 247.144 248.128
      F 166.086 148.076 149.06

Table 2: Modified fragment ions obtained from the StavroX 3.6.0.1 analysis. Where, ‘b’ and ‘y’ ions for m/z 1404.900 identified by StavroX 3.6.0.1 analysis are shown in Table 3.

Intens. Rel Intens. m/z calc. Dev(Da) Type Z Peptide Loss
4999.0 100.0 202.107 198.113 3.994 y5 +3 α 0
4999.0 100.0 202.107 204.112 -2.005 b2 +4 α 0
2568.0 51.4 123.115 113.318 9.798 y3 +4 α 0
2568.0 51.4 123.115 131.077 -7.962 y4 +4 α 0
2568.0 51.4 123.115 133.081 -9.966 y2 +2 β 0
2226.6 44.5 216.122 221.792 -5.67 y6 +3 α 0
2226.6 44.5 216.122 221.871 -5.749 b3 +4 α 0
2226.6 44.5 216.122 225.628 -9.506 y3 +2 α 0
1611.0 32.2 567.361 570.801 -3.441 b1 +2 β 0
1358.7 27.2 258.168 261.147 -2.978 y4 +2 α 0
1358.7 27.2 258.168 265.155 -6.986 y2 +1 β 0
1285.8 25.7 485.285 478.254 7.03 b4 +2 α 0
1050.0 21.0 341.242 332.184 9.058 y6 +2 α 0
1050.0 21.0 341.242 347.439 -6.196 P0 +4   18
1050.0 21.0 341.242 347.685 -6.442 P0 +4   17
958.0 19.2 285.181 276.39 8.792 b5 +4 α 0
958.0 19.2 285.181 285.904 -0.723 b1 +4 β 0
906.9 18.1 145.048 148.836 -3.788 y5 +4 α 0
906.9 18.1 145.048 150.755 -5.706 y3 +3 α 0
906.9 18.1 145.048 152.111 -7.062 y2 +2 α 0
827.8 16.6 324.217 319.172 5.045 b4 +3 α 0
824.2 16.5 624.421 615.819 8.601 b6 +2 α 0
824.2 16.5 624.421 620.336 4.085 b2 +2 β 0
541.0 10.8 245.149 239.631 5.519 b4 +4 α 0
541.0 10.8 245.149 248.135 -2.986 b1 +3 α 0
476.2 9.5 416.227 407.217 9.01 b2 +2 α 0
476.2 9.5 416.227 410.882 5.345 b6 +3 α 0
476.2 9.5 416.227 413.893 2.334 b2 +3 β 0
452.5 9.1 367.259 368.184 -0.925 b5 +3 α 0
452.5 9.1 367.259 371.698 -4.439 b1 +2 α 0
428.0 8.6 528.326 521.285 7.039 y4 +1 α 0
408.0 8.2 159.063 166.086 -7.023 y1 +1 β 0
408.0 8.2 159.063 166.596 -7.532 y6 +4 α 0
386.0 7.7 301.212 295.493 5.719 b3 +3 α 0
386.0 7.7 301.212 296.665 4.547 y5 +2 α 0
386.0 7.7 301.212 303.214 -2.001 y2 +1 α 0
386.0 7.7 301.212 308.413 -7.201 b6 +4 α 0
386.0 7.7 301.212 310.671 -9.459 b2 +4 β 0
335.2 6.7 459.271 450.249 9.022 y3 +1 α 0
335.2 6.7 459.271 462.916 -3.645 P0 +3   18
335.2 6.7 459.271 463.244 -3.973 P0 +3   17
335.2 6.7 459.271 468.919 -9.648 P0 +3   0
335.0 6.7 685.383 693.87 -8.487 P0 +2   18
335.0 6.7 685.383 694.362 -8.979 P0 +2   17
263.0 5.3 385.24 380.87 4.37 b1 +3 β 0
263.0 5.3 595.408 592.324 3.085 y5 +1 α 0
238.4 4.8 699.397 702.875 -3.478 P0 +2   0
180.0 3.6 811.551 813.427 -1.876 b2 +1 α 0

Table 3: The ‘b’ and ‘y’ ions for m/z 1404.900 identified by StavroX 3.6.0.1 analysis.

An intermolecularly crosslinked peptide fragment m/z 1404.900 is shown in Figure 5. It contains Peptide 1 (α-peptide) “KVF” crosslinked to peptide 2 (β-peptide) “LAAAmKR”, which shows that intermolecular cross-linking has occurred between K1 (Lysine-1 in the primary structure of Lysozyme) of the α-peptide with L8 (Leucine-8 in the primary structure of Lysozyme) of the β-peptide of another moiety of Lysozyme. This is shown as a representative example from many such intermolecular crosslinks established by StavroX 3.6.0.1.

analytical-bioanalytical-techniques-identified

Figure 5: The ‘b’ and ‘y’ ions identified by StavroX 3.6.0.1 analysis.

PyMol software

Using the software PyMol, 3D (SI-XIII) representation of the intermolecular cross-link between the two Lysozyme molecules as obtained by using our new heterobifunctional cross-linker and StavroX 3.6.0.1 analysis is depicted in Figure 6.

analytical-bioanalytical-techniques-lysozyme

Figure 6: Two Lysozyme molecules intermolecularly crosslinked drawn using PyMol software.

Crosslinking studies on Lysozyme using homobifunctional crosslinkers was discussed in a seminal and highly cited paper by A. Sinz’s group [3]. Cross-linking reactions with sulfo-DST and sulfo- EGS yielded no cross-linking products, while the cross-linking reaction with BS3 gave two cross-linking products. The details of the nature of this cross-linking (mostly intramolecular cross-linking) are included in Table 4, reproduced from this earlier work.

Cross-linking reagent Cross-linking product Observed mass Sequence (amino acid)
BS3 N-Terminal-K1 744.442 1-5+XL
  K96-K97 1643.868 87-100+Xl

Table 4:Earlier reported Identification of crosslinking of Lysozyme with the homo bifunctional cross-linker, the di-NHS ester, BS3.

In addition, our experiments also identified many intermolecular cross-linking in MS and ESI-MS based crosslinked peptide not detected by the earlier workers. In comparison, our experiments have led to enhanced intermolecular cross-linking (Table 5).

Cross-linking reagent Cross-linking product Observed mass Sequence (amino acid)
CXL-379.0935 N-Terminal of S-R S 100, R 114 1383.523 100-114, 90-120 + XL
  K1-C115 1378.778 1-120 + XL
  K96-R114 1383.830 90-120 + XL
  K96-A110 1212.763 95-120 + XL
  T118-G16 2311.145 110-30 + XL
  Q50-Y20 1912.146 40+30 + XL

Table 5: Intermolecular cross-links identified by StavroX 3.6.0.1 with our new heterobifunctional cross-linker.

Our results thus justify the hypothesis originally put up by Hagan Bayley [loc.cit.] based on perflourophenylazides and extended by us to our aryl azides, which do not require ortho-flanking fluorines, as in the case of perflourophenylazides. Aryl azides that lead to ‘long-lived’ transients bring about more efficient intermolecular cross-linking, which is the case with our new hetero-bi-functional cross-linker. As stated earlier, this observation can have an impact on diverse areas of science.

Conclusions

A new small aryl-azido-NHS-hetero-bifunctional cross-linker has been synthesized and characterized spectroscopically. It has been successfully used to crosslink Lysozyme as a ‘proof of concept’. This is done in two steps, i.e., via an initial incubation step, which is then followed by the second step of photolysis (366 nm) using a 6 W TLC visualization UV lamp. It was then subjected to SDS-PAGE, excision of the ‘dimeric’ band, trypsin digestion, desalting using ziptip and analysis by ESI-MS. The data thus obtained was fed into the StavroX 3.6.0.1, a bioinformatics tool, especially suited for studies on intermolecular crosslinking, which established the crosslinking sites.

The above study confirms that the new crosslinker successfully brings about intermolecular crosslinking and that the use of ESI-MS/ MS/MS along with StavroX 3.6.0.1, the bioinformatics software, greatly facilitates the analysis of intermolecular crosslinking of the two protein molecules.

The above technique has implications in diverse fields, e.g., for studies on protein-protein interactions, for proteomics/lipidomics and in systems and structural biology. It is expected to help in preparing monoclonal antibody-drug conjugates, which specifically target tumor cells representing “the pinnacle of such targeting efforts.” Recently it has been shown that combining cryo-electron microscopy (cryo-EM) with chemical crosslinking will pave the way for highly efficient in vivo studies. The technique is also important in many areas of materials science.

Supplementary Information (SI)

A new Heterobifunctional cross-linker has been characterized spectroscopically its molecular formula has been established based on its observed [M+ H+] m/z 379.0935 calculated value for [M+ H+] m/z 378.299 The new cross-linker has amine reactive N-Hydroxysuccinimide and a photo-reactive aryl azide group. It has been used to cross-linked two lysozyme molecules which was proved by the appearance of ‘Dimeric band’ in the SDS-PAGE. This band was subjected to Trypsin digestion, ESI-MS and StravroX 3.6.0.1 the bio-informatics software helped establish the crosslinking sites. The, study has implication in proteinprotein interaction and diverse areas of science.

Acknowledgements

Authors thank Dr Sudhanshu Vrati, Executive Director, Regional Centre for Biotechnology (RCB) Faridabad, Haryana for research facilities and AcSIR for Emeritus Professoship (Hony.) (to SVE). We thank the Advanced Technology Platform Center (ATPC, RCB) and Dr Nirpender Singh (ESI-MS, MS/MS studies) for his advice and foir supervising the MS studies. We are grateful to Dr David Cane, Brown University, Providence, Rhode Island, USA (FAB- MS); Prof Vani Bramhachari, Ambedkar Centre for Biomedical Research, University of Delhi, Delhi (initial SDS-PAGE work); Dr Andrea Sinz, University of Halle-Salle, Germany (for helpful discussions) and Dr Michael Goetze from the same group (for StavroX 3. 6.0.1 software and its use).

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Citation: Thakur KS, Eswaran SV (2018) ESI-MS and Stavrox 3.6.0.1 Investigations of Crosslinking by an Aryl-Azido-NHS-Heterobifunctional Crosslinker. J Anal Bioanal Tech 9: 402. DOI: 10.4172/2155-9872.1000402

Copyright: © 2018 Thakur KS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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