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Extract msn

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Extract-msn is a versatile and reliable lab equipment product designed for the extraction of biomolecules. It utilizes a robust and efficient extraction process to isolate a wide range of target analytes, including proteins, nucleic acids, and small molecules, from various sample types. The core function of Extract-msn is to provide researchers and scientists with a streamlined and reproducible method for sample preparation prior to downstream analysis.

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Lab products found in correlation

Bioworks 3, by Thermo Fisher Scientific (6 mentions) Mascot daemon, by Matrix Science (3 mentions) Mascot v2, by Matrix Science (3 mentions) Mascot server, by Matrix Science (1 mentions) Bioworks cluster 3, by Thermo Fisher Scientific (1 mentions) Mascot, by Matrix Science (1 mentions) Mascot version 2, by Matrix Science (1 mentions) Sequest software, by Thermo Fisher Scientific (1 mentions) Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)

12 protocols using extract msn

1

Tandem Mass Spectrometry Protocol for Protein Identification

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Tandem mass spectra were extracted using extract_msn (Thermo Fisher Scientific) executed in Mascot Daemon (version 2.2.2; Matrix Science). Peak list files were searched against the IPI Human database (version 3.70, release date 4 March 2010) modified to contain ten additional contaminants and reagent sequences of non-human origin. Searches were submitted to an in-house Mascot server (version 2.2.03; Matrix Science)55 (link) as described previously7 (link). Mass tolerances for precursor and fragment ions were 0.4 and 0.5 Da, respectively. Data were validated in Scaffold (version 3.00.06; Proteome Software) using a threshold of identification of at least 90% probability at the peptide level, assignment of at least two unique, validated peptides, and at least 99% probability at the protein level (estimated protein false discovery rate 0.1% for all data sets).
Byron A., Askari J.A., Humphries J.D., Jacquemet G., Koper E.J., Warwood S., Choi C.K., Stroud M.J., Chen C.S., Knight D, & Humphries M.J. (2015). A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting. Nature Communications, 6, 6135.
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2

Intact Protein Identification from MS/MS

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LC-MS/MS raw data were converted into “.dta” files using Extract_MSn (version 3.0) from Bioworks Cluster 3.2 (Thermo Scientific). MS/MS spectra were identified based on database searching against a human protein database (UniprotKB, released 2010–05) using both SEQUEST (version 27, revision 12) and MS-Align + algorithms [26 (link)]. MS-Align + is an algorithm designed for intact protein identification and is capable of identifying high mass peptides from MS/MS data. Filtering criteria using either MS-Generating Function Score (MS-GF) <1E-10 or MS-Align false discovery rate (FDR) <0.05 were applied to ensure the FDR of final peptide identifications were below 1% based on a decoy database searching strategy [27 (link)].
Sigdel T.K., Nicora C.D., Hsieh S.C., Dai H., Qian W.J., Camp DG I.I, & Sarwal M.M. (2014). Optimization for peptide sample preparation for urine peptidomics. Clinical proteomics, 11(1), 7.
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3

Mass Spectrometry-based Protein Identification

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Tandem mass spectra were extracted using extract_msn (Thermo Fisher Scientific) executed in Mascot Daemon (version 2.4; Matrix Science). Peak list files were searched against a modified version of the Uniprot mouse database (version 3.70; release date, 3 May 2011), containing ten additional contaminant and reagent sequences of non-mouse origin, using Mascot (version 2.2.06; Matrix Science) (Perkins et al, 1999). Carbamidomethylation of cysteine was set as a fixed modification; oxidation of methionine and hydroxylation of proline and lysine were allowed as variable modifications. Only tryptic peptides were considered, with up to one missed cleavage permitted. Monoisotopic precursor mass values were used, and only doubly and triply charged precursor ions were considered. Mass tolerances for precursor and fragment ions were 0.4 Da and 0.5 Da, respectively. MS datasets were validated using rigorous statistical algorithms at both the peptide and protein level45 (link),46 (link) implemented in Scaffold (version 3.6.5; Proteome Software). Protein identifications were accepted upon assignment of at least two unique validated peptides with ≥90% probability, resulting in ≥99% probability at the protein level. These acceptance criteria resulted in an estimated protein false discovery rate of 0.1% for all datasets.
Randles M.J., Lausecker F., Humphries J.D., Byron A., Clark S.J., Miner J.H., Zent R., Humphries M.J, & Lennon R. (2020). Basement membrane ligands initiate distinct signalling networks to direct cell shape. Matrix Biology, 90, 61-78.
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4

Proteomic Analysis of Human Samples

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Mass spectra were extracted using extract_msn (Thermo Fisher Scientific) correlated against the Uniprot human database (87 (link)) using Mascot version 2.5.1 (Matrix Science, London, UK).
Search parameters were as follows: species, Homo sapiens; enzyme, trypsin for SMARTTM-digested samples and nonspecific for elastase-digested samples; maximum missed cleavages, 1; fixed modifications, carbamidomethyl (mass, 57.02; AA, C); variable modification, oxidation (mass, 15.99; AA, M); peptide tolerance, 10 ppm (monoisotopic); fragment tolerance, 0.6 Da (monoisotopic); searched database, SwissProt_2016_04 (152,544 protein entries).
Data generated were validated using Scaffold (Proteome Software; Portland, OR). Only exclusive, unique peptide counts are reported. False discovery rate (FDR) was calculated by Scaffold using protein and peptide probabilities assigned by the Trans-Proteomic Pipeline and the Protein ProphetTM (88 (link)) and Peptide ProphetTM (89 (link)) algorithm (Sourceforge, Seattle, WA). Peptide Prophet FDR was thresholded to ≤5%, and Protein Prophet FDR was thresholded to ≤0.1% (minimum of 2 peptides) for every data set.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (90 (link)) partner repository with the data set identifier PXD008450 and 10.6019/PXD008450.
Eckersley A., Mellody K.T., Pilkington S., Griffiths C.E., Watson R.E., O'Cualain R., Baldock C., Knight D, & Sherratt M.J. (2018). Structural and compositional diversity of fibrillin microfibrils in human tissues. The Journal of Biological Chemistry, 293(14), 5117-5133.
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5

Mass Spectrometry Analysis of PMF-G

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Picomole quantities of PMF-G were provided to the University of Louisville Biomolecular Mass Spectrometry Core Laboratory. Intact protein mass was determined by electrospray ionisation mass spectrometry (ESI-MS) using a Q-TOF API-US (Waters Division, Milford, MA), while proteolytic fragment fingerprints were acquired by liquid chromatography tandem mass spectroscopy (LC/MS-MS) using a LTQ Orbitrap XL (Thermo Scientific, Waltham, MA). SEQUEST software (Thermo Scientific, Waltham, MA), MassMatrix v.1.3.2 [34] (link), or custom Python scripts built around Extract-MSn (Thermo Scientific, Waltham, MA) were used for all peptide analyses. The average masses of intact proteins and monoisotopic masses of peptides were matched to theoretical average or monisotopic masses, respectively. Predicted intact masses were adjusted by 1.0078 Da per cysteine to account for the protons displaced in disulfide bonds.
Wilburn D.B., Bowen K.E., Doty K.A., Arumugam S., Lane A.N., Feldhoff P.W, & Feldhoff R.C. (2014). Structural Insights into the Evolution of a Sexy Protein: Novel Topology and Restricted Backbone Flexibility in a Hypervariable Pheromone from the Red-Legged Salamander, Plethodon shermani. PLoS ONE, 9(5), e96975.
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6

Quantitative Proteomics by LC-MS/MS

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Peak lists were generated using extract-msn as part of Bioworks 3.3.1 (Thermo Scientific) using the following parameters: minimum mass 300; maximum mass 5,000; grouping tolerance 0.01 Da; intermediate scans 200; minimum group count 1; 10 peaks minimum and total ion current of 100. Peak lists for each LC-MS/MS run were merged into a single mascot generic format. Automatic charge state recognition was used because of the high resolution survey scan (30,000). LC-MS/MS spectra were searched against the NCBI RefSeq human protein database [55 (link)] in a target decoy fashion using X!Tandem Sledgehammer (2013.09.01.1). Search parameters used were: fixed modification (carboamidomethylation of cysteine; +57 Da), variable modifications (oxidation of methionine; +16 Da and N-terminal acetylation; +42 Da), three missed tryptic cleavages, 20 ppm peptide mass tolerance and 0.6 Da fragment ion mass tolerance. Protein identifications with at least 2 unique peptides were shortlisted to obtain a master list with less than 0.5% false discovery rate.
Keerthikumar S., Gangoda L., Liem M., Fonseka P., Atukorala I., Ozcitti C., Mechler A., Adda C.G., Ang C.S, & Mathivanan S. (2015). Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes. Oncotarget, 6(17), 15375-15396.
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7

Peptide Identification by MASCOT Search

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Peak lists were generated using extract-msn as part of Bioworks 3.3.1 (Thermo Scientific) using the following parameters: minimum mass 300, maximum mass 5,000; grouping tolerance 0.01 Da; intermediate scans 200; minimum group count 1; 10 peaks minimum and total ion current of 100. Peak lists for each LC-MS/MS run were merged into a single mascot generic format. Automatic charge state recognition was used because of the high-resolution survey scan (30,000). LC-MS/MS spectra were searched against the NCBI RefSeq database in a target decoy manner using MASCOT (v2.4, Matrix Science, UK). Search parameters used were as follows: fixed modification (carboamidomethylation of cysteine; +57 Da), variable modifications (oxidation of methionine; +16 Da), three missed tryptic cleavages, 20 p.p.m. peptide mass tolerance and 0.6 Da fragment ion mass tolerance. Peptide identifications with mascot ion score greater than the identity score were deemed significant. With a cut-off of two unique peptides per protein, <0.5% false discovery rate was achieved.
Atkin-Smith G.K., Tixeira R., Paone S., Mathivanan S., Collins C., Liem M., Goodall K.J., Ravichandran K.S., Hulett M.D, & Poon I.K. (2015). A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure. Nature Communications, 6, 7439.
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8

Bovine Protein Identification via LC-MS/MS

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Peak lists were generated using extract-msn as part of Bioworks 3.3.1 (Thermo Scientific) using the following parameters: minimum mass 300; maximum mass 5,000; grouping tolerance 0.01 Da; intermediate scans 200; minimum group count 1; 10 peaks minimum and total ion current of 100. Peak lists for each LC-MS/MS run were merged into a single mascot generic format. Automatic charge state recognition was used because of the high resolution survey scan (30,000). LC-MS/MS spectra were searched against the NCBI RefSeq bovine protein database28 (link) using X!Tandem Sledgehammer (2013.09.01.1). Search parameters used were: fixed modification (carboamidomethylation of cysteine; +57 Da), variable modifications (oxidation of methionine; +16 Da and N-terminal acetylation; +42 Da), three missed tryptic cleavages, 20 ppm peptide mass tolerance and 0.6 Da fragment ion mass tolerance.
Samuel M., Chisanga D., Liem M., Keerthikumar S., Anand S., Ang C.S., Adda C.G., Versteegen E., Jois M, & Mathivanan S. (2017). Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth. Scientific Reports, 7, 5933.
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9

Robust Peptide Identification via High-Resolution LC-MS/MS

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Extract-MSn as part of Bioworks 3.3.1 (Thermo Scientific) was used to generate peak list files with the following parameters: minimum mass 300; maximum mass 5,000; grouping tolerance 0 Da; intermediate scans 200; minimum group count 1; 10 peaks minimum and total ion current of 100. Peak lists for LC-MS/MS runs were merged into a single mascot generic file format. Automatic charge state recognition was used due to the high-resolution survey scan (30,000). LC-MS/MS spectra were searched against the NCBI RefSeq human protein database in a target decoy fashion using X!Tandem Sledgehammer (2013.09.01.1). Searching parameters used were: fixed modification (carboamidomethylation of cysteine; +57 Da), variable modifications (oxidation of methionine; +16 Da) and N-terminal acetylation; +42 Da), three missed tryptic cleavages, 20 ppm peptide mass tolerance and 0.6 Da fragment ion mass tolerance. Protein identifications with at least 2 unique peptides were shortlisted and false discovery rate was less than 0.5% [22].
Kalra H., Gangoda L., Fonseka P., Chitti S.V., Liem M., Keerthikumar S., Samuel M., Boukouris S., Al Saffar H., Collins C., Adda C.G., Ang C.S, & Mathivanan S. (2019). Extracellular vesicles containing oncogenic mutant β-catenin activate Wnt signalling pathway in the recipient cells. Journal of Extracellular Vesicles, 8(1), 1690217.
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10

Protein Identification by LC-MS/MS in Plasmodium falciparum

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For protein identification of protein spots LC-MS/MS data were searched against a non-redundant protein decoy database comprising sequences from P. falciparum, as well as their reverse sequences and common contaminants. Mass spectra peak lists were extracted using extract-msn as part of Bioworks 3.3.1 (Thermo Fisher Scientific) linked into Mascot Daemon (Matrix Science, UK). Peak lists for each nano-LC-MS/MS run were searched using MASCOT v2.2.04 (Matrix Science, UK), provided by the Australian Proteomics Computational Facility (www.apcf.edu.au). The search parameters consisted of carbamidomethylation of cysteine as a fixed modification (+57 Da), with variable modifications set for NH2-terminal acetylation (+42 Da) and oxidation of methionine (+16 Da). A precursor mass tolerance of 20 ppm, fragment ion mass tolerance of ±0.04 Da and an allowance for up to three missed cleavages for tryptic searches was used. Scaffold v4.4.3 (Proteome Software, Inc., USA) was used to validate protein identifications derived from MS/MS sequencing results (FDR <0.5%).
Sisquella X., Nebl T., Thompson J.K., Whitehead L., Malpede B.M., Salinas N.D., Rogers K., Tolia N.H., Fleig A., O’Neill J., Tham W.H., David Horgen F, & Cowman A.F. (2017). Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion. eLife, 6, e21083.
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