Computational protocol: Carbamoylation correlates of cyanate neuropathy and cyanide poisoning: relevance to the biomarkers of cassava cyanogenesis and motor system toxicity

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Protocol publication

[…] Ten μg samples of peptide digests were injected onto a trap cartridge (Michrom Bioresources, Auburn, CA) at 20 μl/min, samples were washed for 5 min with 2% acetonitrile, 0.1% formic acid, and the trap cartridge placed in-line with a 0.5 × 250 mm SB-C18 reverse phase column (Agilent Technologies, Santa Clara, CA). Peptides were then eluted using a 200 min gradient of 7-30% acetonitrile containing 0.1% formic acid at a flow rate of 10 μl/min. Peptide m/z values (MS spectra) and fragment ions (MS/MS spectra) were collected using an LTQ linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA) with an ion max electrospray source fitted with a 34Ga metal needle. Each survey MS spectrum from 400–2000 m/z was followed by 3 data-dependant MS/MS spectra on the 3 most intense ions found in each survey MS spectrum. The instrument used the dynamic exclusion feature of its control software to ignore previously analyzed ions (repeat count of 1, maximum exclusion list of 50, and 30 sec exclusion time), a tune file configured with one μscan, a maximum ion accumulation time of 200 m/sec, and AGC targets of 30,000 and 10,000, respectively, for MS and MS/MS spectra.MS/MS spectra from each analysis were converted to dta files using Bioworks 3.3 (Thermo Scientific) and each MS/MS spectrum matched to peptide sequences in a database using the program Sequest (Version 27, rev. 12, Thermo Scientific). Searches were performed with a static modification of +57 on cysteines for carboxyimidomethylation, and differential modifications of +16 for oxidation of methionines, and +43 for carbamoylation of lysines. The search also used trypsin specificity. A database of rat sequences was created from the Uniprot database (Swiss Bioinformatics Institute, Geneva, Switzerland) downloaded in Feb. 2011 (15,524 entries), the sequences of common contaminants added, and the database amended with sequence reversed entries to estimate peptide false discovery rates. Sequest results were then processed using the program Scaffold (version 4.0.5, Proteome Software, Portland, OR) to produce a list of identified peptides and their carbamoylation sites. Peptide and protein confidence levels of 95% and 99% were used, and a minimum of 2 unique peptides matching each protein entry was required.Since few carbamoylated peptides were detected in the soluble fraction of spinal cord, further analysis to test for differences in the level of carbamoylation between different treatment groups was only performed for serum and spinal cord insoluble fractions. Differences in levels of carbamoylation in various treatment groups was determined by tabulating the numbers of MS/MS spectra assigned to carbamoylated peptides from albumin in serum digests, and the proteins myelin basic protein, neurofilament light polypeptide, 2’3’-cyclic-nucleotide 3’-phosphodiesterase, myelin proteolipid protein, and glial fibrillary acidic protein in digests of water-insoluble proteins from spinal cord. Inclusion lists for each of the identified carbamolyated peptides was created by specifying the m/z value for each in the instrument’s control software. This maximized the collection of MS/MS spectra for carbamoylated peptides during the quantitative analysis so that a shorter data collection period was required. This mass spectrometric analysis was identical to those described above. However they used a 60 min gradient, inclusion lists specifying the m/z values of the targeted carbamoylated peptides, and the dynamic exclusion feature of the instrument’s control software was disabled. Differences in the level of carbamoylation in various sample groups was then calculated by summing the numbers of MS/MS scans (spectral counts) assigned to carbamoylated peptides from the target proteins in each sample. The MS/MS scans collected during the analysis were again searched using Sequest using the same database and differential searches for modified peptides as before. However, due to the size of the result files, the Sequest results were filtered using PAW software (Wilmarth et al. ), which controlled peptide false discovery using numbers of matches to the sequence reversed entries. Peptides identified as being carbamoylated were separately filtered from unmodified peptides to maintain their false discovery rate below 5%. The linearity of the carbamoylation detection was tested by mixing protein digests from cyanate and vehicle treated animals from the sulfur deficient diet group (Figure ). The numbers of identified carbamoylated peptides as a function of percent cyanate treated serum or spinal cord protein was determined following Sequest searches and filtering of MS/MS data using Scaffold as described above.Figure 2 […]

Pipeline specifications

Software tools MSCAN, Comet
Application MS-based untargeted proteomics
Organisms Manihot esculenta, Rattus norvegicus
Diseases Amino Acid Metabolism, Inborn Errors, Multiple Sclerosis, Nervous System Diseases, Poisoning, Drug-Related Side Effects and Adverse Reactions
Chemicals Amino Acids, Sulfur