scholarly journals Ultraviolet Photodissociation of tryptic peptide backbones at 213 nm

2020 ◽  
Author(s):  
Lars Kolbowski ◽  
Adam Belsom ◽  
Juri Rappsilber
Keyword(s):  
Reference Beam ◽  
Aromatic Amino Acids ◽  
Sequence Coverage ◽  
E Coli ◽  
Sequence Fragment ◽  
Ultraviolet Photodissociation ◽  
Fragmentation Behavior ◽  
Excitation Time ◽  
Beam Type ◽  
Supplemental Activation

We analyzed the backbone fragmentation behavior of tryptic peptides of a four protein mixture and of E. coli lysate subjected to Ultraviolet Photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15,178 high-confidence peptide-spectrum matches by additionally recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor. Type a, b and y ions were most prominent in UVPD spectra and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity) and remaining precursor intensity high. Sequence coverage and sequence fragment intensity ratio correlated with precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to non-covalent interactions. UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points at aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange with identifier PXD018176 and on spectrumviewer.org/db/UVPD_213nm_trypPep.

scholarly journals Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Amino Acids ◽  
2021 ◽  
Author(s):  
Magdalena Widgren Sandberg ◽  
Jakob Bunkenborg ◽  
Stine Thyssen ◽  
Martin Villadsen ◽  
Thomas Kofoed
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Peptide Mapping ◽  
Covalent Modification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
E Coli ◽  
Gm Csf ◽  
Fragmentation Behavior ◽  
Macrophage Colony

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

Enhanced Sequence Coverage of Large Proteins by Combining Ultraviolet Photodissociation with Proton Transfer Reactions

2019 ◽  
Vol 92 (1) ◽  
pp. 1041-1049 ◽  
Author(s):  
James D. Sanders ◽  
Christopher Mullen ◽  
Eleanor Watts ◽  
Dustin D. Holden ◽  
John E. P. Syka ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Transfer Reactions ◽  
Sequence Coverage ◽  
Large Proteins ◽  
Ultraviolet Photodissociation ◽  
Proton Transfer Reactions

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals The Analysis of OmpA and Rz/Rz1 of Lytic Bacteriophage from Surabaya, Indonesia

Scientifica ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Tessa Sjahriani ◽  
Eddy Bagus Wasito ◽  
Wiwiek Tyasningsih
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Statistical Tests ◽  
Lytic Bacteriophage ◽  
Ligand Binding Site ◽  
Sequence Coverage ◽  
E Coli ◽  
Food Borne ◽  
Peptide Composition ◽  
Partial Genome

A good strategy to conquer the Escherichia coli-cause food-borne disease could be bacteriophages. Porins are a type of β-barrel proteins with diffuse channels and OmpA, which has a role in hydrophilic transport, is the most frequent porin in E. coli; it was also chosen as the potential receptor of the phage. And the Rz/Rz1 was engaged in the breakup of the host bacterial external membrane. This study aimed to analyze the amino acid of OmpA and Rz/Rz1 of lytic bacteriophage from Surabaya, Indonesia. This study employed a sample of 8 bacteriophages from the previous study. The OmpA analysis method was mass spectrometry. Rz/Rz1 was analyzed using PCR, DNA sequencing, Expasy Translation, and Expasy ProtParam. The result obtained 10% to 29% sequence coverage of OmpA, carrying the ligand-binding site. The Rz/Rz1 gene shares a high percentage of 97.04% to 98.89% identities with the Siphoviridae isolate ctTwQ4, partial genome, and Myoviridae isolate cthRA4, partial genome. The Mann–Whitney statistical tests indicate the significant differences between Alanine, Aspartate, Glycine, Proline, Serine ( p = 0.011 ), Asparagine, Cysteine ( p = 0.009 ), Isoleucine ( p = 0.043 ), Lysine ( p = 0.034 ), Methionine ( p = 0.001 ), Threonine ( p = 0.018 ), and Tryptophan ( p = 0.007 ) of OmpA and Rz/Rz1. The conclusion obtained from this study is the fact that OmpA acts as Phage 1, Phage 2, Phage 3, Phage 5, and Phage 6 receptors for its peptide composition comprising the ligand binding site, and Rz/Rz1 participates in host bacteria lysis.

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.

scholarly journals Tyrosine Aminotransferase Catalyzes the Final Step of Methionine Recycling in Klebsiella pneumoniae

1999 ◽  
Vol 181 (6) ◽  
pp. 1739-1747 ◽  
Author(s):  
Jacqueline Heilbronn ◽  
Judith Wilson ◽  
Bradley J. Berger
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Amino Acid Sequence ◽  
Paracoccus Denitrificans ◽  
Rhizobium Meliloti ◽  
Aromatic Amino Acids ◽  
Tyrosine Aminotransferase ◽  
E Coli ◽  
Substrate Analogues ◽  
Terminal Amino

ABSTRACT An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of α-ketomethiobutyrate to methionine, has been purified fromKlebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, andRhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with α-ketomethiobutyrate and of glutamate with α-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with α-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.

scholarly journals Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics

2019 ◽  
Vol 19 (2) ◽  
pp. 405-420 ◽  
Author(s):  
Luca Fornelli ◽  
Kristina Srzentić ◽  
Timothy K. Toby ◽  
Peter F. Doubleday ◽  
Romain Huguet ◽  
...  
Keyword(s):  
High Throughput ◽  
Ad Hoc ◽  
Laser Pulses ◽  
Sequence Coverage ◽  
Top Down ◽  
Inference Problem ◽  
Ultraviolet Photodissociation ◽  
Matching Strategy ◽  
Dissociation Mechanisms ◽  
High Degree

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.

Adaptability of nonnatural aromatic amino acids to the active center of the E. coli ribosomal A site

FEBS Letters ◽  
1993 ◽  
Vol 335 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Takahiro Hohsaka ◽  
Ken Sato ◽  
Masahiko Sisido ◽  
Kazuyuki Takai ◽  
Shigeyuki Yokoyama
Keyword(s):  
Amino Acids ◽  
Active Center ◽  
Aromatic Amino Acids ◽  
E Coli ◽  
A Site

Comparison of the UV Resonance Raman Spectra of Bacteria, Bacterial Cell Walls, and Ribosomes Excited in the Deep UV

1993 ◽  
Vol 47 (1) ◽  
pp. 38-43 ◽  
Author(s):  
S. Chadha ◽  
R. Manoharan ◽  
P. Moënne-Loccoz ◽  
W. H. Nelson ◽  
W. L. Peticolas ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Raman Spectra ◽  
Resonance Raman ◽  
Aromatic Amino Acids ◽  
Enterobacter Aerogenes ◽  
Large Intensity ◽  
Whole Cells ◽  
E Coli ◽  
Bacterial Rna ◽  
Resonance Raman Spectra

Resonance Raman spectra have been obtained with 218-nm excitation for Escherichia coli and Staphylococcus epidermidis. Large intensity differences seen in the tryptophan-associated 1556-cm−1 peak appear to be strongly related to Gram type. Unlike E. coli, S. epidermidis possess a very intense peak at 1658 cm−1 which varies in intensity with cultural conditions. Spectra excited from E. coli and Enterobacter aerogenes with 200-nm light show peaks which strongly reflect nucleic acid composition, unlike spectra excited at 218 nm. Purified, separated ribosomes of E. coli produce spectra which are dominated by nucleic acid vibrations when excited at 242 nm, but have peaks belonging nearly exclusively to protein aromatic amino acids when excited at 222 nm. The relative weakness of bacterial RNA modes excited at 222 nm from whole cells and ribosomes is attributed to nucleic acid hypochromism.

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