Trypsin digested peptides were dissolved in 0.1% (v/v) TFA and mixed with equal volume of matrix (50% v/v acetonitrile,0.1% v/v TFA and 20 gL− 1 α-cyano-4-hydroxycinnamic acid in ultrapure water). The peptide-matrix solution was loaded on to a MALDI plate and allowed to dry. MS/MS analysis was performed on a MALDI-TOF/TOF MS analyzer (AB-SCIEX, TOF/TOF 5800, Applied Biosystems, USA). The peptide mass spectra were detected through result-dependent analysis on Protein Pilot™ v.3.2 software (AB Sciex, MA, USA) with MS (precursor-ion) peak filtering 800–4000 m/z interval, monoisotopic, mass tolerance 50 ppm. MS/MS (fragmentation) peak filtering monoisotopic, MH+, minimum signal-to-noise ratio (S/N) 10, MS/MS fragment tolerance 0.75 Da. Protein identification was done using MASCOT database search engine (Matrix Science, London, UK) with MS/MS ion search performed against non-redundant NCBI protein database. Parameters were set as taxonomy-viridiplantae, enzyme-trypsin with one missed cleavage, fixed modification to carbamidomethyl C, peptide and MS/MS tolerance set to 1 Da and 0.8 Da respectively with 2+ peptide charge. Significant hits with MASCOT probability-based score (p < 0.05) and best matched molecular weight, pI value and percent sequence coverage were considered to evaluate protein identification.
Mascot database search engine
Manufactured by Matrix Science
Sourced in United Kingdom
MASCOT is a database search engine used for the identification of proteins from mass spectrometry data. It compares the experimental mass spectra against theoretical spectra generated from protein sequence databases.
Automatically generated - may contain errors
Lab products found in correlation
Absciex tof tof 5800, by Thermo Fisher Scientific (1 mentions)
Q tof premier mass spectrometer, by Waters Corporation (1 mentions)
Ltq velos pro, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rapid separation lc, by Thermo Fisher Scientific (1 mentions)
Beh c18 analytical column, by Waters Corporation (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
11 protocols using mascot database search engine
1
MALDI-TOF/TOF MS Analysis of Tryptic Peptides
2
Protein Identification by Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify the purified protein, the band obtained from SDS-PAGE was excised, the in-gel was digested with trypsin, and analyzed by ESI-QUAD-TOF instrument (Cheng et al., 2014 (link)). The detected peptide sequence data were performed automatically by database matching against NCBInr protein database (NCBI) using MASCOT database search engine (Matrix Science; version 2.1.1.0; www.matrixscience.com ). The search parameters were set as follows: trypsin digestion with one missed tryptic cleavage site, methionines carbamidomethyl for cysteines, and oxidation for methionines as variable modifications. The sequence peptides with high matching score were aligned by using CLUSTALX software (version 2.0) (Larkin et al., 2007 (link)).
3
Protein Identification via MASCOT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spectra were processed using flexAnalysis software. Monoisotopic peptide masses were assigned and used in the database search. The protein identification was accomplished utilizing the MASCOT database search engine (Matrix Science, London, UK) (http://www.matrixscience.com ). Probability-based MW search scores were estimated by comparison of search results against an estimated random match population and were reported as 10 log10 (P), where P is the absolute probability. Scores >63 were shown to be significant (P<0.05) in the Mascot search. Proteins identified with scores less than the significant level were reported as unidentified.
4
Purification and Identification of TEM8-Fc Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fusion protein consisting of the N-terminal 1–227 amino acid residues of human TEM8 linked to the Fc portion of human IgG1 (TEM8-Fc) was produced, as described previously [4 (link)]. Human hepatoma tissue was homogenized in RIPA buffer and centrifuged. The supernatant was applied to a protein A Sepharose 4 Fast Flow affinity column to remove endogenous IgG proteins and was transferred to a protein A Sepharose 4 Fast Flow affinity column pre-conjugated with the TEM8-Fc. Bound proteins were eluted and separated by SDS-PAGE, and then stained by Coomassie. A single distinct band of approximately 50 kDa was observed. This band was excised from the gel, incubated with trypsin, and then analyzed by nano LC-MS/MS using CapLC liquid chromatography coupled to a Q-TOF Ultima fusion quadrupole time-of-flight mass spectrometer (Waters, Milford, MA, USA). MS/MS fragment ion spectra were searched against the NCBI nr protein sequence database using the MASCOT database search engine (Matrix Science, London, UK).
5
Protein Identification via MASCOT Database
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein identification was accomplished utilizing the MASCOT database search engine (v.2.3.02, Matrix Science, London). The MS/MS spectra were used to search the IPI human 3.74 database in which trypsin and up to one miscleavage were specified. Carbamidomethylation (C) was set as a fixed modification, while oxidation (M) and deamination (N) were set as variable modifications. A peptide tolerance of 20 ppm and a tolerance of ± 0.7 Da for the fragment ions were used. The peptide identification was filtered by a Mascot Score above 30 with p < 0.05. N-Glycopeptide identification were filtered by a deamidated (N) site at N-X-S/T motif (X is any amino acid except Pro) to reduce potential false positive identification [53 (link)].
6
Peptide Identification from Mass Spectra
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mass spectra were extracted using the Proteome Discoverer (version 2.5). All MS/MS samples were analyzed using the Mascot database search engine (version 2.6; Matrix Science, London, UK). Mascot was set up to search for an in-house database with the digestion enzyme none specific. Mascot was searched using a product ion mass tolerance of 0.60 Da and a precursor ion tolerance of 5.0 ppm by considerations of sensitivity, acquisition time, and the goal of obtaining more candidates through a more relaxed database search filter. Gln->pyro-Glu at the N-terminus, oxidation of methionine and acetyl at the N-terminus, and phospho-serine, threonine, and tyrosine were specified in Mascot as variable modifications. Scaffold (version 4, Proteome Software, https://www.proteomesoftware.com/ ) was used to validate the MS/MS-based peptide and protein identification. To include a broader range of peptide matches, a relatively low threshold of 10 was set for the Mascot ion scores of candidate peptides. To preserve the possibility of identifying additional false negatives, FDR-based cutoff was not employed to further adjust the Mascot search results [20] . Candidate sequences (CandiSeqs) were identified based on the following criteria: 1) maximum Mascot ion score (MaxMascotIonScore) higher than 10; 2) presence of mutation points; 3) length of 8–12mers.
7
Characterization of PsbW Fusion Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PsbW protein (GenBank: CAA59409.1) was obtained from GenScript at purity above 85%. Cytochrome b6 as a fusion protein (ss-cytochrome b6) to the C-proximal part of the signal pre-sequence of the Oxygen Evolving Complex (SLQSDFKELAHKCEASKIAGFALA TSALVASGASA, OE33, GenBank: BAA02554.1).
TheAla-X-Ala consensus sequence that is recognised by a thylakoid processing peptidase (TPP) and that cleaves off this transit sequence was changed to protect fusion protein from being cleaved (SLQSDFKELAHKCEASKIAGFALV TSALVASGR SA). An additional Lys was incorporated into C-terminus in order to allow for biotin labelling. The molecular weight of synthetic protein was assessed with the MALDI and ESI-MS techniques (electrospray ionization mass spectroscopy, QTOF Premier mass spectrometer, Waters Corp), using the Mascot database search engine (version 2.1, Matrix Science)56 (link).
The
8
Tandem Mass Spectrometry Protein Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tandem Mass spectrometry (MS/MS) identification of proteins was conducted using the “gel top” method. Briefly, proteins were separated electrophoretically for 10 min at 150 V by SDS-PAGE and then stained using Instant Blue. Bands of interest were excised from the gel and dehydrated using acetonitrile followed by vacuum centrifugation. Dried gel pieces were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide. Gel pieces were then washed alternately with 25 mM ammonium bicarbonate followed by acetonitrile. This was repeated, and the gel pieces dried by vacuum centrifugation. Samples were digested with trypsin overnight at 37°C. Digested samples were analysed by LC–MS/MS using an UltiMate® 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to a LTQ Velos Pro (Thermo Fisher Scientific, Altrincham, United Kingdom) mass spectrometer. Peptide mixtures were separated using a gradient from 92% A (0.1% FA in water) and 8% B (0.1% FA in acetonitrile) to 33% B, in 44 min at 300 nl min−1, using a 75 mm x 250 μm i.d. 1.7 ·M BEH C18, analytical column (Waters). Peptides were selected for fragmentation automatically by data dependant analysis. Data produced were searched using Mascot data base search engine (Matrix Science United Kingdom). Data were validated using Scaffold (Proteome Software, Portland, OR).
9
Proteomic Analysis of Chelidonium majus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stained protein bands were analyzed by liquid chromatography coupled to LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland. Excised gel fragments were placed in 1.5 mL Eppendorf tubes filled with 10% methanol and 2% acetic acid. The proteins were digested using trypsin. The generated peptides were concentrated, desalted on an RP-C18 precolumn (LC Packings, Coventry, UK), and further separated by UltiMate nano-HPLC (LC Packings, San Francisco, CA, USA). The column outlet was directly coupled to a Nanospray ion source operating in a data-dependent MS to MS/MS switch mode. Identification of proteins matching with the C. majus CDS database was performed (Cmajus 20150107_1; 209,790 sequences; 74,516,318 residues) [8 (link),10 (link)] by using the MASCOT database search engine (Matrix Science, London, UK; www.matrixscience.com ; accessed on 28 October 2021).
10
Proteomic Identification via MASCOT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bioinformatics data mining was performed using in-house licensed version of the MASCOT database search engine (Matrix science, Boston, MA). The SWISS-PORT Database searches were carried out using the following parameters: database, Swiss-Prot; taxonomy, Homo sapien; enzyme, trypsin; and allowance of one missed cleavage. Protein that returned MOWSE scores over a threshold of 50 were considered significant. Further analysis and function based classification of identified proteins was performed using the Protein Centre software version 3.10 by Thermo Fisher Scientific.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!