Optimized Fragmentation Improves the Identification of Peptides Cross-Linked by MS-Cleavable Reagents

Abstract

Cross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometrycleavable (MS-cleavable) reagents dramatically reduces complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)-based crosslinks on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped-HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD-method was then directly compared with previously described methods using an Orbitrap Fusion™ Lumos™ Tribrid TM instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS (MS n ) enabled instrumentation and alternative dissociation techniques..

A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes

“…MS2-CID: CID collision energy 30%, MS2 resolution 30K, AGC target 5e4, maximum ion injection time 100 ms. MS2-ETD: MS2 resolution 30K, AGC target 1e5, maximum ion injection time 120 ms. MS3: MS2 isolation window 2m/z, collision energy 35%, AGC target 3e4, maximum ion injection time 90 ms). Settings recommended by Stieger et al 32 were used for stepped HCD methods (MS1 orbitrap resolution 60K, MS1 m/z scan range 375-1500, MS1 maximum injection time 50 ms, MS1 AGC target 5e5, dynamic exclusion 30 s with 10 ppm mass window, charge state exclusion <3+ or >8+, isolation window 1.6m/z, stepped HCD with normalised collision energies 21%, 27% and 33%, TopN N = 10 starting with the most intense MS1 signal, MS2 resolution 30K, MS2 maximum injection time 100 ms, MS2 AGC target 5e4). Fig.…”

Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides

“…Finally we estimated the correctness of the software calculated FDR rate of the obtained data, by separating cross-link hits found within the ribosomal shotgun proteins 9 and found within the human proteome from our searches using the combined database. As shown in Supplemental Figure 6, for XlinkX, the number of "wrong" cross-links from or to human proteins varies between 4 -8 % of the total hit number and is therefore in the expected range.…”

“…We are aware that -by means of obtained absolute XL numbers -others have reported more unique cross-links (up to 766 unique links for E.coli ribosome using DSSO or DSBU and fractionation using SEC or SCX respectively 9,31 ). Enriching via an affinity tag may still be very advantageous in case of a complex matrix, as this would be the case for in vivo investigations.…”

“…Since cross-linked peptides are on average larger and higher charged, compared to linear peptides, enrichment is often done via size exclusion (SEC) [7][8][9][10] or strong cation exchange chromatography (SCX) [11][12][13] , respectively. A bottleneck in cross-linking studies regarding complex systems remains, in that coverage is almost exclusively restricted to the most abundant proteins (e.g.…”

Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin

“…Our group has previously shown that the number of identified peptides, when using alternatives to trypsin, could largely be improved by a sequential combination with trypsin. This includes AspN, GluC, chymotrypsin, and elastase for the detection of cross-link sites 12 − 15 and elastase applied to S. pombe whole cell lysates. 14 The sequential digestion increased the number of identified cross-links up to 19-fold for the Taf4–12 complex compared to digesting with elastase alone.…”