An integrated workflow for crosslinking mass spectrometry

Mendes, Marta; Fischer, Lutz; Chen, Zhuo A.; Barbon, Marta; O’Reilly, Francis J.; Giese, Sven H.; Bohlke-Schneider, Michael; Belsom, Adam; Dau, Therese; Combe, Colin; Graham, Martin; Eisele, Markus R.; Baumeister, Wolfgang; Speck, Christian; Rappsilber, Juri

doi:10.15252/msb.20198994

Cited by 158 publications

(151 citation statements)

References 58 publications

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“…Protein and peptide identifications improved when combining any of the tested proteases with trypsin. This is in line with previous studies on cross-linking identification, which benefited from the sequential digest with trypsin 12,14 . In the most extreme case, the sequential digest with trypsin and AspN outperformed results obtained by trypsin alone.…”

Section: Resultssupporting

confidence: 90%

“…In this way trypsin leads to a concentration increase of peptides by reducing sample complexity. At least when trypsin is used first an additional mechanism must be considered that was previously described for sequential digestion 12,14 . The second enzyme does not cleave shorter peptides with high efficiency effectively leading to short tryptic peptides being protected from proteinase K. In either case, the complexity that is normally introduced through proteinase K is reduced by the tryptic treatment.…”

Section: Resultsmentioning

confidence: 99%

“…Proteinase K for example, was used, to "shave" surface exposed loops from proteins in membrane vesicles 10,11 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 crosslink sites [12][13][14] and elastase applied to S. pombe whole cell lysates 14 . The sequential digestion increased the number of identified crosslinks up to 19-fold for the Taf4-12 complex compared to digesting with elastase alone 14 .…”

Section: Introductionmentioning

confidence: 99%

“…Introducing positively charged C-termini through trypsin improves the detection of previously non-tryptic peptides. Importantly, smaller peptides are protected from the second protease 12,14 . Thus, use of two proteases does not lead to the very small peptides that in silico digestion would predict.…”

Section: Introductionmentioning

confidence: 99%

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

Dau

Bartolomucci

Rappsilber

2020

Preprint

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Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted on the number of proteins identified. Proteases with higher specificity let to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, pre-digesting with trypsin improves the number of identified proteins for proteinase K by 731 %. Trypsin pre-digestion also improved the protein identifications of other proteases, AspN (+62 %), GluC (+80 %) and chymotrypsin (+21 %). Interestingly, the sequential digest with trypsin and AspN yielded even higher number of protein identifications than digesting with trypsin alone.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Dau

Bartolomucci

Rappsilber

2020

Preprint

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Matzinger

Kandioller

Doppler

et al. 2019

Preprint

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ABSTRACTCross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. This results in challenging data evaluation and the need for an effective enrichment.Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose® beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ~100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2 – 1:100. The number of detectable unique cross-links maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 −5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 μg cross-linked E. coli ribosome in a background of 100 μg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced.This robust, fast and selective enrichment method for azide-tagged linkers will contribute to map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.

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Cross‐linking reactions in food proteins and proteomic approaches for their detection

Renzone

Arena

Scaloni

2021

Mass Spectrometry Reviews

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Various protein cross‐linking reactions leading to molecular polymerization and covalent aggregates have been described in processed foods. They are an undesired side effect of processes designed to reduce bacterial load, extend shelf life, and modify technological properties, as well as being an expected result of treatments designed to modify raw material texture and function. Although the formation of these products is known to affect the sensory and technological properties of foods, the corresponding cross‐linking reactions and resulting protein polymers have not yet undergone detailed molecular characterization. This is essential for describing how their generation can be related to food processing conditions and quality parameters. Due to the complex structure of cross‐linked species, bottom‐up proteomic procedures developed to characterize various amino acid modifications associated with food processing conditions currently offer a limited molecular description of bridged peptide structures. Recent progress in cross‐linking mass spectrometry for the topological characterization of protein complexes has facilitated the development of various proteomic methods and bioinformatic tools for unveiling bridged species, which can now also be used for the detailed molecular characterization of polymeric cross‐linked products in processed foods. We here examine their benefits and limitations in terms of evaluating cross‐linked food proteins and propose future scenarios for application in foodomics. They offer potential for understanding the protein cross‐linking formation mechanisms in processed foods, and how the inherent beneficial properties of treated foodstuffs can be preserved or enhanced.

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An integrated workflow for crosslinking mass spectrometry

Cited by 158 publications

References 58 publications

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

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

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

Cross‐linking reactions in food proteins and proteomic approaches for their detection

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