Enhancing Open Modification Searches via a Combined Approach Facilitated by Ursgal

Schulze, Stefan; Igiraneza, Aime Bienfait; Kösters, Manuel; Leufken, Johannes; Leidel, Sebastian A.; García, Benjamin A.; Fufezan, Christian; Pohlschröder, Mechthild

doi:10.1021/acs.jproteome.0c00799

Cited by 9 publications

(7 citation statements)

References 54 publications

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“…On the other hand, the concept of peptidoform has been used in several studies [15–26], but not widely. Peptidoform stands for specifically modified or mutated peptides with the same backbone amino acid sequence [21].…”

Section: Top‐down and Bottom‐up Proteomics: Proteoform And Peptidoformmentioning

confidence: 99%

A peptidoform based proteomic strategy for studying functions of post‐translational modifications

Liu

2021

Proteomics

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Protein post‐translational modifications (PTMs) generate an enormous, but as yet undetermined, expansion of the produced proteoforms. In this Viewpoint, we firstly reviewed the concepts of proteoform and peptidoform. We show that many of the current PTM biological investigation and annotation studies largely follow a PTM site‐specific rather than proteoform‐specific approach. We further illustrate a potentially useful matching strategy in which a particular “modified peptidoform” is matched to the corresponding “unmodified peptidoform” as a reference for the quantitative analysis between samples and conditions. We suggest this strategy has the potential to provide more directly relevant information to learn the PTM site‐specific biological functions. Accordingly, we advocate for the wider use of the nomenclature “peptidoform” in future bottom‐up proteomic studies.

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Section: Top‐down and Bottom‐up Proteomics: Proteoform And Peptidoformmentioning

confidence: 99%

A peptidoform based proteomic strategy for studying functions of post‐translational modifications

Liu

2021

Proteomics

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“…Unfortunately, we could not assess whether peptides containing 2 N -glycosites could be modified with 1 AglB- and 1 Agl15-dependent N -glycan, since the employed protein database search engines do not support the simultaneous search for 2 different modifications of the same amino acid. However, mass shifts corresponding to a combination of AglB- and Agl15-dependent N -glycosylation could not be found in results from an open modification search [ 38 ].…”

Section: Resultsmentioning

confidence: 99%

Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea

et al. 2021

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Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.

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“…(Timp and Timp 2020) Recent advances in open modification search tools in BUP have inspired more studies into underexplored PTMs. (Chick et al, 2015;Kong et al, 2017;Schulze et al, 2021) Our TDP data presented here also took advantage of open modification search for "hypothesis-free" PTM discovery.…”

Section: Discussionmentioning

confidence: 99%

Discovery top-down proteomics in symbiotic soybean root nodules

Zhou

Fulcher²,

Zemaitis³

et al. 2022

Front. Anal. Sci.

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Proteomic methods have been widely used to study proteins in complex biological samples to understand biological molecular mechanisms. Most well-established methods (known as bottom-up proteomics, BUP) employ an enzymatic digestion step to cleave intact proteins into smaller peptides for liquid chromatography (LC) mass spectrometry (MS) detection. In contrast, top-down proteomics (TDP) directly characterizes intact proteins including all possible post-translational modifications (PTMs), thus offering unique insights into proteoform biology where combinations of individual PTMs may play important roles. We performed TDP on soybean root nodules infected by the symbiotic Bradyrhizobium japonicum in both the wildtype bacterium and a nifH- mutant, which lacks the ability to fix nitrogen in the soybean root nodule. TDP captured 1648 proteoforms derived from 313 bacterial genes and 178 soybean genes. Leghemoglobin, the most abundant protein in the sample, existed in many truncated proteoforms. Interestingly, these truncated proteoforms were considerably more abundant in the wildtype relative to the nifH- mutant, implicating protease activity as an important factor in nitrogen fixation. Proteoforms with various PTMs and combinations thereof were identified using an unrestricted open modification search. This included less common PTMs such as myristoylation, palmitoylation, cyanylation, and sulfation. In parallel, we collected high resolution MS imaging (MSI) data of intact proteins and biopolymers (<20 kDa due to current technical limitations) from sections of the soybean root nodules using matrix-assisted laser desorption/ionization (MALDI) coupled to high resolution Orbitrap. Several detected proteoforms exhibited unique spatial distributions inside the infection zone and cortex, suggesting functional compartmentalization in these regions. A subset of peaks from the MALDI-MSI were assigned to proteoforms detected in TDP LCMS data based on matching accurate masses. Many of the proteins detected in both LCMS and MALDI-MSI are currently uncharacterized in UniProt: the PTM and spatial information presented here will be valuable in understanding their biological functions. Taken together, our study demonstrates how untargeted TDP approach can provide unique insights into plant proteoform biology. On-going technology developments are expected to further improve TDP coverage for more comprehensive high-throughput analysis of proteoforms.

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Enhancing Open Modification Searches via a Combined Approach Facilitated by Ursgal

Cited by 9 publications

References 54 publications

A peptidoform based proteomic strategy for studying functions of post‐translational modifications

A peptidoform based proteomic strategy for studying functions of post‐translational modifications

Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea

Discovery top-down proteomics in symbiotic soybean root nodules

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