Mass Accuracy and Sequence Requirements for Protein Database Searching

This study developed a multilayered, gel-based, and underivatized strategy forde novoprotein sequence analysis of unsequenced dinoflagellates using a MALDI-TOF/TOF mass spectrometer with the assistance of DeNovo Explorer software. MASCOT was applied as the first layer screen to identify either known or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed after searching against the NCBInr database, the remainder was searched against the dinoflagellate expressed sequence tag database. In the last layer, those borderline and nonconfident hits were further subjected tode novointerpretation using DeNovo Explorer software. Thede novosequences passing a reliability filter were subsequently submitted to nonredundant MS-BLAST search. Using this layer identification method, 216 protein spots representing 158 unique proteins out of 220 selected protein spots fromAlexandrium tamarense, a dinoflagellate with unsequenced genome, were confidently or tentatively identified by database searching. These proteins were involved in various intracellular physiological activities. This study is the first effort to develop a completely automated approach to identify proteins from unsequenced dinoflagellate databases and establishes a preliminary protein database for various physiological studies of dinoflagellates in the future.

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Faculty Opinions recommendation of Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009203.120858 ◽

2002 ◽

Author(s):

Samuel Gunderson

Keyword(s):

Length Regulation ◽

Sequence Requirements

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Faculty Opinions recommendation of Sub-2 microm HPLC coupled with sub-ppm mass accuracy for analysis of pharmaceutical compound libraries.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1133852.591944 ◽

2008 ◽

Author(s):

Christopher Cimarusti

Keyword(s):

Mass Accuracy ◽

Pharmaceutical Compound ◽

Compound Libraries

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Faculty Opinions recommendation of Systematic mutagenesis of α-synuclein reveals distinct sequence requirements for physiological and pathological activities.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717961178.793464433 ◽

2012 ◽

Author(s):

Robert Petersen

Keyword(s):

Distinct Sequence ◽

Sequence Requirements

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Mass Spectrometry-Based Identification of Urinary Antimicrobial Peptides in Dairy Cows

Protein and Peptide Letters ◽

10.2174/0929866526666191025105038 ◽

2020 ◽

Vol 27 (3) ◽

pp. 225-235

Author(s):

Ambika Sharma ◽

Rajesh Nigam ◽

Ashish Kumar ◽

Simmi Singh

Keyword(s):

Mass Spectrometry ◽

Antimicrobial Peptides ◽

Polyacrylamide Gel Electrophoresis ◽

Radial Diffusion ◽

Protein Database ◽

Antimicrobial Effects ◽

Cow Urine ◽

Radial Diffusion Assay ◽

Promising Source ◽

Diffusion Assay

Background:: Urine is considered one of the biological fluids in which antimicrobial peptides are secreted or expressed. Cow urine has not been investigated for the presence of these peptides using MALDI-TOF-MS. Objective:: The aim of this study is to isolate, identify and assess the antimicrobial activity of urinary antimicrobial peptides from healthy normal cycling cows. Method:: We analyzed the urine sample using diafiltration, ion exchange chromatography, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), acid urea polyacrylamide gel electrophoresis (AU-PAGE) coupled with identification through Peptide Mass Fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF- MS). The in vitro antimicrobial effects of purified fractions were assessed using Radial Diffusion Assay (RDA) and microtitre broth dilution assay against Gram-positive and Gramnegative bacteria. Results: : Proteins corresponding to the peaks were identified using SWISSPROT protein database. This study revealed constitutive expression of β-Defensin-1 (DEFB1), β-Defensin-4A (DFB4A), Neutrophil Defensin-1 (DEF1), Neutrophil Defensin-3 (DEF3) in cow urine. The identified peptides are cationic antimicrobial peptides of the defensin family. The purified fractions exhibited antimicrobial effects in radial diffusion assay and MIC values in the range of 2.93-29.3 &*#181;M/L. Conclusion:: This study concludes that cow urine, previously unexplored with regard to antimicrobial peptides, would be a promising source of highly potent AMPs and an effective alternative to the resistant antibiotics.

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