scholarly journals A General Protease Digestion Procedure for Optimal Protein Sequence Coverage and Post-Translational Modifications Analysis of Recombinant Glycoproteins: Application to the Characterization of Human Lysyl Oxidase-like 2 Glycosylation

2011 ◽  
Vol 83 (22) ◽  
pp. 8484-8491 ◽  
Author(s):  
Kathryn R. Rebecchi ◽  
Eden P. Go ◽  
Li Xu ◽  
Carrie L. Woodin ◽  
Minae Mure ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Lysyl Oxidase ◽  
Sequence Coverage ◽  
Post Translational Modifications ◽  
Digestion Procedure ◽  
Recombinant Glycoproteins ◽  
Protease Digestion ◽  
Protein Sequence Coverage

scholarly journals Increased Single-Spectrum Top-Down Protein Sequence Coverage in Trapping Mass Spectrometers with Chimeric Ion Loading

2020 ◽  
Vol 92 (18) ◽  
pp. 12193-12200
Author(s):  
Chad R. Weisbrod ◽  
Lissa C. Anderson ◽  
Joseph B. Greer ◽  
Caroline J. DeHart ◽  
Christopher L. Hendrickson
Keyword(s):  
Protein Sequence ◽  
Sequence Coverage ◽  
Top Down ◽  
Single Spectrum ◽  
Mass Spectrometers ◽  
Protein Sequence Coverage

scholarly journals 2018 YPIC Challenge: A case study in characterizing an unknown protein sample

2019 ◽  
Author(s):  
Lindsay Pino ◽  
Andy Lin ◽  
Wout Bittremieux
Keyword(s):  
Protein Sequence ◽  
De Novo ◽  
Signal To Noise Ratio ◽  
3D Structure ◽  
Sequence Information ◽  
Sequence Coverage ◽  
Post Translational Modifications ◽  
E Coli ◽  
Unknown Protein ◽  
Synthetic Protein

For the 2018 YPIC Challenge contestants were invited to try to decipher two unknown English questions encoded by a synthetic protein expressed in Escherichia coli. In addition to deciphering the sentence, contestants were asked to determine the 3D structure and detect any post-translation modifications left by the host organism. We present our experimental and computational strategy to characterize this sample by identifying the unknown protein sequence and detecting the presence of post-translational modifications. The sample was acquired with dynamic exclusion disabled to increase the signal-to-noise ratio of the measured molecules, after which spectral clustering was used to generate high-quality consensus spectra. De novo spectrum identification was used to determine the synthetic protein sequence, and any post-translational modifications introduced by E. coli on the synthetic protein were analyzed via spectral networking. This workflow resulted in a de novo sequence coverage of 70%, on par with sequence database searching performance. Additionally, the spectral networking analysis indicated that no systematic modifications were introduced on the synthetic protein by E. coli. The strategy presented here can be directly used to analyze samples for which no protein sequence information is available or when the identity of the sample is unknown. All software and code to perform the bioinformatics analysis is available as open source, and self-contained Jupyter notebooks are provided to fully recreate the analysis.

Citraconylation—a simple method for high protein sequence coverage in MALDI-TOF mass spectrometry

2003 ◽  
Vol 305 (4) ◽  
pp. 1091-1093 ◽  
Author(s):  
Vojtěch Kadlčı́k ◽  
Martin Strohalm ◽  
Milan Kodı́ček
Keyword(s):  
Mass Spectrometry ◽  
Protein Sequence ◽  
High Protein ◽  
Sequence Coverage ◽  
Simple Method ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Protein Sequence Coverage

Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery

PROTEOMICS ◽  
2009 ◽  
Vol 9 (3) ◽  
pp. 783-787 ◽  
Author(s):  
Haruhiko Kamada ◽  
Tim Fugmann ◽  
Dario Neri ◽  
Christoph Roesli
Keyword(s):  
Protein Sequence ◽  
Biomarker Discovery ◽  
Cysteine Modification ◽  
Sequence Coverage ◽  
Protein Sequence Coverage

scholarly journals Effects of Guanidination with Trypsin, Lys-C, or Glu-C Digestion on Mass Spectrometric Signal Intensity and Protein Sequence Coverage

2010 ◽  
Vol 31 (6) ◽  
pp. 1527-1534 ◽  
Author(s):  
Hye-Sun Han ◽  
Seon-Ho Nho ◽  
Ae-Ra Lee ◽  
Jeong-Kwon Kim
Keyword(s):  
Protein Sequence ◽  
Signal Intensity ◽  
Mass Spectrometric ◽  
Sequence Coverage ◽  
Protein Sequence Coverage

scholarly journals In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

2021 ◽  
Author(s):  
Luis Ariel Espinosa ◽  
Yassel Ramos ◽  
Ivan Andujar ◽  
Enso Onill Torres ◽  
Gleysin Cabrera ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Expression Systems ◽  
Sequence Coverage ◽  
Subunit Vaccines ◽  
Post Translational Modifications ◽  
Esi Ms ◽  
Intermolecular Disulfide Bond ◽  
Protein Primary Structure ◽  
Very High

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

100% protein sequence coverage: a modern form of surrealism in proteomics

Amino Acids ◽  
2010 ◽  
Vol 41 (2) ◽  
pp. 291-310 ◽  
Author(s):  
Bjoern Meyer ◽  
Dimitrios G. Papasotiriou ◽  
Michael Karas
Keyword(s):  
Protein Sequence ◽  
Sequence Coverage ◽  
Modern Form ◽  
Protein Sequence Coverage

scholarly journals Data-independent acquisition protease-multiplexing enables increased proteome sequence coverage across multiple fragmentation modes

2021 ◽  
Author(s):  
Alicia L Richards ◽  
Kuei-Ho Chen ◽  
Damien B Wilburn ◽  
Erica Stevenson ◽  
Benjamin Polacco ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Routine Data ◽  
Resonance Excitation ◽  
Sequence Coverage ◽  
Data Independent Acquisition ◽  
Beam Type ◽  
Quantitative Performance ◽  
Mode Beam ◽  
Time Required ◽  
Protein Sequence Coverage

The use of multiple proteases has been shown to increase protein sequence coverage in proteomics experiments, however due to the additional analysis time required, it has not been widely adapted in routine data-dependent acquisition (DDA) proteomic workflows. Alternatively, data-independent acquisition (DIA) has the potential to analyze multiplexed samples from different protease digests, but has been primarily optimized for fragmenting tryptic peptides. Here we evaluate a DIA multiplexing approach that combines three proteolytic digests (Trypsin, AspN, and GluC) into a single sample. We first optimize data acquisition conditions for each protease individually with both the canonical DIA fragmentation mode (beam type CID), as well as resonance excitation CID, to determine optimal consensus conditions across proteases. Next, we demonstrate that application of these conditions to a protease-multiplexed sample of human peptides results in similar protein identifications and quantitative performance as compared to trypsin alone, but enables up to a 63% increase in peptide detections, and a 27% increase non-redundant amino acid detections. Importantly, this resulted in 100% sequence coverage for numerous proteins, suggesting the utility of this approach in applications where sequence coverage is critical, such as proteoform analysis.

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