X tandem

Manufactured by Matrix Science

Sourced in United Kingdom

The X! Tandem is a mass spectrometry software tool designed for protein identification and characterization. It performs database searches to match experimental tandem mass spectrometry data with theoretical peptide spectra generated from protein sequence databases.

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Lab products found in correlation

Mascot, by Matrix Science (8 mentions) Ltq linear ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)

8 protocols using x tandem

Venom Proteomic Profiling of Three Species

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Analyses by reversed-phased nano chromatography coupled to tandem mass spectrometry analyses of the venoms from three species were performed by the Florida State University College of Medicine Translational laboratory and by the Laboratory of Toxinology (FIOCRUZ, Rio de Janeiro), as detailed in the supplementary methods, Supplementary material online. Protein identifications of the obtained spectra were performed using MASCOT (Matrix Science, London, UK; version 2.6.2) and X! Tandem (The GPM, thegpm.org, last accessed August 3, 2020; version X! Tandem Alanine [2017.2.1.4]) as the search engine. We considered a 99% and 95% threshold for protein and peptide identification, respectively. Custom-generated FASTA databases containing curated sequences of identified toxins for each specimen and translated protein sequences from the assembled transcriptome (Trinity contigs) for the species were used as a database for spectral identification, as detailed in the supplementary methods, Supplementary material online. To quantify the estimated abundance of each toxin class, we normalized the total spectra of all identified proteins using the Normalized Spectral Abundance Factor (NSAF) as implemented in Scaffold 5 (Zybailov et al. 2006 (link)).

Bayona-Serrano J.D., Grazziotin F.G., Salazar-Valenzuela D., Valente R.H., Nachtigall P.G., Colombini M., Moura-da-Silva A, & Junqueira-de-Azevedo I.L. (2023). Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae). Molecular Biology and Evolution, 40(7), msad147.

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Proteomic analysis of human samples

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All MS/MS samples were analyzed using Mascot (Matrix Science; version 2.2.04) and X! Tandem (The GPM, thegpm.org; version CYCLONE [2010.12.01.1]). Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. The UniprotHuman_2013_08 database (88,378 entries) was searched. Carbamidomethyl of cysteine was specified as a fixed modification and Glu->pyro-Glu of the n-terminus, ammonia-loss of the n-terminus, gln->pyro-Glu of the n-terminus, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine were specified in X! Tandem as variable modifications. Oxidation of methionine and phospho of serine, threonine, and tyrosine were specified in Mascot as variable modifications.

Divorty N., Jenkins L., Ganguly A., Butcher A.J., Hudson B.D., Schulz S., Tobin A.B., Nicklin S.A, & Milligan G. (2022). Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor. The Journal of Biological Chemistry, 298(3), 101655.

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Protein Identification using Tandem Mass Spectrometry

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Tandem mass spectra were extracted by ProteoWizard version 3.0.7529 from the raw files. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.02) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)). Mascot was set up to search the uniprot_pig_20150721 database (selected for Mammalia, unknown version, 26153 entries) assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the uniprot_sprot_20150223 database also assuming trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine was specified in Mascot and X! Tandem as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine, acetyl of the n-terminus and phospho of serine, threonine and tyrosine were specified in Mascot as variable modifications. Glu->pyro-Glu of the n-terminus, ammonia-loss of the n-terminus, gln->pyro-Glu of the n-terminus, deamidated of asparagine and glutamine, oxidation of methionine, acetyl of the n-terminus, carbamidomethyl of cysteine and phospho of serine, threonine and tyrosine were specified in X! Tandem as variable modifications.

Paresi C.J., Liu Q, & Li Y.M. (2016). Benzimidazole covalent probes and the gastric H+/K+-ATPase as a model system for protein labeling in a copper-free setting. Molecular bioSystems, 12(6), 1772-1780.

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Proteomics Analysis of S. cerevisiae

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Tandem mass spectra were extracted using ProteoWizard MsConvert. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Science; version 2.4.0) and X! Tandem (The GPM, thegpm.org; version X! Tandem Sledgehammer (2013.09.01.1)). Mascot was set up to search the S. cerevisiae Swissprot database (downloaded in April 2019, 16,060 entries), assuming the digestion enzyme stricttrypsin. X! Tandem was set up to search the S. cerevisiae Swissprot database (downloaded in April 2019, 16,060 entries) also assuming stricttrypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified in Mascot and X! Tandem as a fixed modification. Glu->pyro-Glu of the N-terminus, ammonia-loss of the N-terminus, Gln->pyro-Glu of the N-terminus, and oxidation of methionine were specified in X! Tandem as variable modifications. Oxidation of methionine and acetyl of the N-terminus were specified in Mascot as variable modifications.

Du Truong C., Craig T.A., Cui G., Botuyan M.V., Serkasevich R.A., Chan K.Y., Mer G., Chiu P.L, & Kumar R. (2021). Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ. The Journal of Biological Chemistry, 297(2), 100912.

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Mass Spectrometry-Based Protein Identification

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All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.02) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)). Mascot was set up to search the Drosophila NCBI protein database (downloaded 2010; 14,335 entries). X! Tandem was set up to search a subset of the Drosophila NCBI protein database assuming the digestion enzyme trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 10.0 PPM for single-step immunopurification. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 2.0 Da for tandem-step immunopurification. Iodoacetamide derivative of cysteine was specified in Mascot and X! Tandem as a fixed modification. Methylation of lysine, oxidation of methionine and phosphorylation of serine, threonine and tyrosine were specified in X! Tandem as variable modifications. Methylation of lysine, oxidation of methionine, acetaldehyde +28 of lysine, formylation of lysine, acetylation of lysine, tri-methylation and di-methylation of lysine and phosphorylation of serine, threonine and tyrosine were specified in Mascot as variable modifications. Variable modifications were accepted if they could be established at greater than 95.0% probability by Mascot.

Swenson J.M., Colmenares S.U., Strom A.R., Costes S.V, & Karpen G.H. (2016). The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic. eLife, 5, e16096.

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Mass Spectrometry Protein Identification Protocol

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All MS/MS samples were analyzed using Mascot (Matrix Science; version 2.2.04) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1). Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. The UniprotHuman_2013_08 database (88,378 entries) and a custom database containing the amino acid sequence of human GPR84-eYFP were searched. Carbamidomethyl of cysteine was specified as a fixed modification, Glu->pyro-Glu of the N terminus, ammonia loss of the N terminus, gln->pyro-Glu of the N terminus, oxidation of methionine and phosphorylation of serine, threonine, and tyrosine were specified in X! Tandem as variable modifications. Oxidation of methionine and phosphorylation of serine, threonine, and tyrosine were specified in Mascot as variable modifications.

Marsango S., Ward R.J., Jenkins L., Butcher A.J., Al Mahmud Z., Dwomoh L., Nagel F., Schulz S., Tikhonova I.G., Tobin A.B, & Milligan G. (2022). Selective phosphorylation of threonine residues defines GPR84–arrestin interactions of biased ligands. The Journal of Biological Chemistry, 298(5), 101932.

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Mass Spectrometry-based Protein Identification

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Tandem mass spectra were extracted by Proteo wizard version 3.0.9.134. Charge state deconvolution and deisotoping were performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.02) and X! Tandem [The GPM, thegpm.org; version CYCLONE (2010.12.01.1)]. Mascot and X! Tandem were set up to search Human, Mus, or MTB databases {150821_SPROT_Human_Iso_AUP000005640 database (unknown version, 91618 entries), 151218_SPROT_MTB_UP000001584 database (unknown version, 3993 entries), or 150612_Mus_uniprot_proteome_AUP000000589 database (unknown version, 45182 entries). Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine was specified in Mascot and X! Tandem as a fixed modification. Deamidation of asparagine and glutamine; oxidation of methionine; formyl of the N terminus; acetyl of lysine; phospho of serine, threonine, and tyrosine; and Gly-Gly of lysine were specified in Mascot as variable modifications. Glu->pyro-Glu of the N terminus; ammonia-loss of the N terminus; gln->pyro-Glu of the N terminus; deamidation of asparagine and glutamine; oxidation of methionine; formyl of the N terminus; acetyl of lysine; phospho of serine, threonine, and tyrosine; and Gly-Gly of lysine were specified in X! Tandem as variable modifications.

Rigo M.M., Borges T.J., Lang B.J., Murshid A., N.i.t.i.k.a., Wolfgeher D., Calderwood S.K., Truman A.W, & Bonorino C. (2020). Host expression system modulates recombinant Hsp70 activity through post-translational modifications. The FEBS journal.

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Identification of Protein of Interest

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Two hundred micrograms of a RuBisCO-depleted leaf extract was loaded on each of two separate 2D SDS-PAGE gels with a first dimension pH range of 3-6 (Supplemental Figure S3, A andB). One gel was visualized by silver staining and the second gel was transferred to a PVDF membrane for blotting with the α-pAdi3 antibody (Supplemental Figure S3A). The two images of the stained gel and WB were overlaid in order to confirm the precise location(s) of the protein of interest on the silver-stained gel. The proteins on the silverstained gel aligning with the WB (Supplemental Figure S3B) were excised for trypsin digestion followed by LC-MS/MS analysis to identify the protein. LC-MS/MS analysis was carried out at the University of Texas, San Antonio mass spectrometry core facility. The samples were reduced and alkylated with iodoacetamide prior to trypsin digestion, and mass spectra of trypsin-digested peptides were obtained using a ThermoFisher LTQ linear ion trap mass spectrometer with nano-LC peptide separations. MS/MS results were analyzed using Mascot (Matrix Science, London, UK; version 2.6.2) and X! Tandem (version CYCLONE). Mascot was set up to search the SwissProt_2017_02 database (553,655 entries) assuming trypsin digestion.

Yeo I.C., de Azevedo Manhaes A.M.E., Liu J., Avila J., He P, & Devarenne T.P. (2023). An unexpected role for tomato threonine deaminase 2 in host defense against bacterial infection. Plant physiology, 192(1).

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