Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase

Taouatas, Nadia; Drugan, Mădălina M.; Heck, Albert J. R.; Mohammed, Shabaz

doi:10.1038/nmeth.1204

Cited by 118 publications

(156 citation statements)

References 14 publications

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“…Intrinsic to the ETD process is the loss of one proton through charge reduction. We have previously demonstrated that in a single proton scenario, the basic moieties influence which fragment ions will be observed (19,21). The proportion of c and z ions is dependent on the basicity present at each terminus.…”

Section: Resultsmentioning

confidence: 99%

“…Using Lys-N, the majority of the proteolytic peptides contain a single N-terminal lysine residue and can be enriched by strong cation exchange (SCX) chromatography (20). These peptides generate predominantly sequence diagnostic "c" ion series upon ETD (19), resulting in easy to interpret sequence ladders, which opens up a unique avenue for de novo sequencing and, as we show here, markedly reduces the dependence on databases.…”

mentioning

confidence: 97%

“…To reduce misassignments, approaches have been suggested based on improved mass accuracy or precision (14,15), combining complementary fragmentation data (14,16,17) or the simplification of fragmentation spectra by using chemical derivatization to manipulate peptide behavior (18). Recently, we introduced a biochemical approach to simplify fragment spectra based on the protease Lys-N (19), which has enzymatic cleavage specificity for the N-terminal side of lysine residues. Using Lys-N, the majority of the proteolytic peptides contain a single N-terminal lysine residue and can be enriched by strong cation exchange (SCX) chromatography (20).…”

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confidence: 99%

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Database independent proteomics analysis of the ostrich and human proteome

Altelaar

Navarro²,

Boekhorst³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Mass spectrometry (MS)-based proteome analysis relies heavily on the presence of complete protein databases. Such a strategy is extremely powerful, albeit not adequate in the analysis of unpredicted postgenome events, such as posttranslational modifications, which exponentially increase the search space. Therefore, it is of interest to explore "database-free" approaches. Here, we sampled the ostrich and human proteomes with a method facilitating de novo sequencing, utilizing the protease Lys-N in combination with electron transfer dissociation. By implementing several validation steps, including the combined use of collision-induced dissociation/ electron transfer dissociation data and a cross-validation with conventional database search strategies, we identified approximately 2,500 unique de novo peptide sequences from the ostrich sample with over 900 peptides generating full backbone sequence coverage. This dataset allowed the appropriate positioning of ostrich in the evolutionary tree. The described database-free sequencing approach is generically applicable and has great potential in important proteomics applications such as in the analysis of variable parts of endogenous antibodies or proteins modified by a plethora of complex posttranslational modifications.

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Section: Resultsmentioning

confidence: 99%

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confidence: 97%

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confidence: 99%

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Database independent proteomics analysis of the ostrich and human proteome

Altelaar

Navarro²,

Boekhorst³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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show abstract

“…This approach would be valuable to distinguish modification by ubiquitin-like proteins (Ubls) versus conventional ubiquitination based on different C-terminal sequences. Also, proteolytic digests using Lys-N, which yield almost complete c-ion series in ETD, could be well suited for this purpose [44]. Finally, multiple-reactionmonitoring (MRM) [45] and "middle-down" mass spectrometry [46] are now also applied to the detection of protein ubiquitination, both of which could potentially benefit from being combined with ETD.…”

Section: Discussionmentioning

confidence: 99%

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

Sobott

Watt

Smith

et al. 2009

J. Am. Soc. Mass Spectrom.

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Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N( ) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular zϩ1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins. (J Am Soc Mass

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“…Furthermore, > 2 + peptide charge states during MS ionization frequently require a higher number of basic amino acids within the respective peptide. Therefore, alternate proteases such as Lys-C, Lys-N, or Arg-C have to be used (Taouatas et al , 2008 ), which nevertheless suffer from reduced specifi city and sequence coverage. Even disregarding further drawbacks such as lower fragmentation effi cacy in comparison to CID and prolonged fragmentation Figure 1 (A) Glycoprotein analysis can be divided into three tasks: elucidation of attachment sites (glycoproteomics), glycan profi ling and structural analysis (glycomics), and fi nally characterization of microheterogeneities.…”

Section: Techniques In Ms-based Glycosylation Site Assignmentmentioning

confidence: 99%

N-glycoproteomics: mass spectrometry-based glycosylation site annotation

Pasing

Sickmann²,

Lewandrowski

2012

BCHM

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Glycosylations are ubiquitous and, in many cases, essential protein modifi cations. Yet comprehensive and detailed analysis of glycosylations on a proteome-wide scale is a daunting and still unsolved challenge. However, a common workfl ow has emerged over the last decade for large-scale N-glycosylation site annotation by application of proteomic methodology. Thereby, the qualitative and quantitative assessment of hundreds or thousands of modifi cation sites is enabled. This review presents a short overview about common enrichment techniques and glycosylation site detection for N-glycopeptides, including benefi ts and challenges of analysis.

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Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase

Cited by 118 publications

References 14 publications

Database independent proteomics analysis of the ostrich and human proteome

Database independent proteomics analysis of the ostrich and human proteome

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

N-glycoproteomics: mass spectrometry-based glycosylation site annotation

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