Spectrum mill ms proteomics workbench

Manufactured by Agilent Technologies

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The Spectrum Mill MS Proteomics Workbench is a software tool designed for the analysis of mass spectrometry data. It provides a comprehensive suite of tools for protein identification, quantification, and data management.

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17 protocols using spectrum mill ms proteomics workbench

SARS-CoV-2 Viral Protein Identification

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For
the viral protein identification, peptide spectral data was extracted,
filtered, and searched against UniProtKB/Swiss-Prot SARS-CoV-2 database (downloaded September 2020). The database contained 14
SARS-CoV-2 proteins. The search was set in Spectrum Mill MS Proteomics
Workbench (Agilent G2721AA/G2733AA) using an automated workflow to
search for peptide sequence matches (scores defined in more detail
in the Supporting Information). Briefly,
protein hits were filtered using peptide database match score >6,
and %SPI (scored peak intensity) >70% (Table S1). We then manually filtered all peptides to include only
peptides
with a false discovery rate (FDR) <1%, which was determined from
a decoy reverse database search (Table S2).

Lara-Jacobo L.R., Islam G., Desaulniers J.P., Kirkwood A.E, & Simmons D.B. (2022). Detection of SARS-CoV-2 Proteins in Wastewater Samples by Mass Spectrometry. Environmental Science & Technology, 56(8), 5062-5070.

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Maize Protein Identification by nLC-nESI-MS/MS

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The bands excised from the non-denaturing Deriphat-PAGE were individually analyzed by tandem mass spectrometry (nLC-nESI-MS/MS) using an 6520 Q-TOF mass spectrometer with HPLC Chip Cube source driven by 1200 series nano/capillary LC system (Agilent Technologies) as previously described (Prinsi and Espen, 2015 (link)). The spectra interpretation was performed by Spectrum Mill MS Proteomics Workbench (Rev B.04.00.127; Agilent Technologies). Cysteine carbamidomethylation and methionine oxidation were set as fixed and variable modifications, respectively, accepting two missed cleavages per peptide. The search was conducted against the subset of Zea mays protein sequences (ID tax: 4577, Oct 2015, 212069 entries) downloaded from the National Center for Biotechnology Information¹ and oncatenated with the reverse one. The threshold used for peptide identification was Spectrum Mill score ≥9, Score Peak Intensity ≥50%, mass MH+ Error ≤±10 ppm, Database Fwd-Rev Score ≥2, and Local False Discovery Rate ≤5%. Protein identification was accepted if confirmed by at least two distinct peptides.

Pii Y., Alessandrini M., Dall’Osto L., Guardini K., Prinsi B., Espen L., Zamboni A, & Varanini Z. (2016). Time-Resolved Investigation of Molecular Components Involved in the Induction of High Affinity Transport System in Maize Roots. Frontiers in Plant Science, 7, 1657.

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Comprehensive Proteomic Data Analysis

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Data were searched using the Spectrum Mill MS Proteomics Workbench (Agilent, Rev B.06.00.201) and the SwissProt human proteome database. For data extraction, spectrum merging was enabled based on precursor selection purity, spectral similarity, retention time (2-minute window), and m/z. Search settings were as follows: instrument = Agilent ESI Q-TOF; precursor mass tolerance = +/- 10 ppm; product ion mass tolerance = +/- 20 ppm; digest = no enzyme. Matches were considered valid if the following thresholds were satisfied: score > 10, percentage scored peak intensity (SPI) > 70%, and rank 1 minus rank 2 (R1-R2) score > 2.5. Spectra that could not be confidently matched to unmodified peptides in the SwissProt database were subjected to a second round of searches using the settings listed above, except oxidized methionine and deamidated asparagine/glutamine were considered as variable modifications and digest was set to AspN (maximum of two missed cleavages). Spectra that remained unmatched according to the validation filters listed above were searched against a custom database containing hypothetical human HIPs (2 (link)) using the same settings as in the first search.

Wiles T.A., Hohenstein A., Landry L.G., Dang M., Powell R., Guyer P., James E.A., Nakayama M., Haskins K., Delong T, & Baker R.L. (2021). Characterization of Human CD4 T Cells Specific for a C-Peptide/C-Peptide Hybrid Insulin Peptide. Frontiers in Immunology, 12, 668680.

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MS-Based Protein Quantification and Purity Analysis

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MS-MS spectra were processed using the Spectrum Mill MS Proteomics Workbench (version A.03.03, Agilent) and filtered MS-MS spectra data were searched against the NCBI database (retrieved on 26 April 2013) for protein identification. Precursor mass tolerance was set at ±20 ppm. One missed cleavage site was allowed in sequence matches. All protein identifications with a protein score ≥ 25 and a peptide SPI ≥ 60%, or a protein score ≥ 15 with no less than two unique peptides identified, were considered as positive identifications. Total peptide ion intensities corresponding to each protein were used for relative protein quantification. Protein abundance was normalized by global intensity using equation (1) as described by Griffin et al47 (link). Additionally, the thylakoid membrane protein purity test, based on the above mentioned protein quantification method, was also conducted. where,
SI_GI is protein abundance normalized to global intensity
SI is protein abundance
SI_j is abundance of the jth protein
n is the number of identified proteins

Gu W., Li H., Zhao P., Yu R., Pan G., Gao S., Xie X., Huang A., He L, & Wang G. (2014). Quantitative proteomic analysis of thylakoid from two microalgae (Haematococcus pluvialis and Dunaliella salina) reveals two different high light-responsive strategies. Scientific Reports, 4, 6661.

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Quantitative Proteomics using HPLC-QTOF

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An HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) connected to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used in this study. Parameters for the equipment analysis were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev. B.08.00, Santa Clara, CA, USA).
Dry samples from trypsin digestion were resuspended in a buffer with water/acetonitrile/formic acid and injected onto an Agilent AdvanceBio Peptide Mapping HPLC column, thermostated at 50 °C, at a flow rate of 0.4 mL/min.
The data processing and protein identification was made on Spectrum Mill MS Proteomics Workbench (Rev B.06.00.201, Agilent Technologies, Santa Clara, CA, USA). The criteria used for MS/MS search against the appropriate and updated protein database were: variable modifications search mode (carbamidomethylated cysteines, STY phosphorylation, oxidized methionine, and N-terminal glutamine conversion to pyroglutamic acid); tryptic digestion with 5 maximum missed cleavages; ESI-Q-TOF instrument (Agilent Technologies, Santa Clara, CA, USA); minimum matched peak intensity 50%; maximum ambiguous precursor charge +5; monoisotopic masses; peptide precursor mass tolerance 20 ppm; product ion mass tolerance 50 ppm; and calculation of reversed database scores.

Rodrigues M., López-Martinez M.J., Ortin-Bustillo A., Cerón J.J., Martinez-Subiela S., Muñoz-Prieto A, & Lamy E. (2023). Changes in the Saliva Proteome of Pigs with Diarrhoea Caused by Escherichia coli. Proteomes, 11(2), 14.

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Mass Spectrometry Analysis of Zona Pellucida

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Proteomics was performed on 50 grown oocytes/group recovered by slicing ovaries in PBS. ZP was solubilized by incubation at 75°C for 45 min (Izquierdo-Rico et al., 2009 (link)) and analyses were carried out on one technical replicate with a HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler module and a High Speed Binary Pump, and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface. Experimental parameters for HPLC and Q-TOF were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev. B.08.00). Data processing and analysis was performed on Spectrum Mill MS Proteomics Workbench (Rev B.06.00.201, Agilent Technologies, Santa Clara, CA, USA).

Lamas-Toranzo I., Fonseca Balvís N., Querejeta-Fernández A., Izquierdo-Rico M.J., González-Brusi L., Lorenzo P.L., García-Rebollar P., Avilés M, & Bermejo-Álvarez P. (2019). ZP4 confers structural properties to the zona pellucida essential for embryo development. eLife, 8, e48904.

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Proteomic Identification of Human Proteins

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The collected cut-out piece from the silver-stained gel was trypsinized in a trypsin digestion buffer (10 mM CaCl₂, 100 mM ammonium bicarbonate, pH 7.8) overnight at 37 °C. The digested sample was subjected to protein identification using a nano-flow liquid chromatography-mass spectrometry apparatus (Agilent 6330 Ion Trap; Agilent Technologies, Santa Clara, CA) equipped with an analytical chip (Agilent HPLC-Chip; Agilent Technologies). The resulting tandem mass spectrometry spectra of the tryptic peptides were analyzed using Agilent software (Spectrum Mill MS Proteomics Workbench; Agilent Technologies) with the protein database (SwissProt) for putative Homo sapiens protein identifications.

Gohara Y., Tomonobu N., Kinoshita R., Futami J., Audebert L., Chen Y., Komalasari N.L., Jiang F., Yoshizawa C., Murata H., Yamamoto K.I., Watanabe M., Kumon H, & Sakaguchi M. (2023). Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells. Journal of Molecular Medicine (Berlin, Germany), 101(4), 431-447.

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Quantitative Proteomic Analysis of Green Algae

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Total soluble protein was extracted from 0.75 mm disks cultured for 0, 24 and 48 h using the phenol method as Wang et al.35 (link) described35 (link). Protein concentration was determined using the Bradford method36 (link). Protein solubilized in 8 M urea was reduced with 10 mM DTT at 37 °C for 1 h and alkylated with 50 mM iodoacetamide in the dark at 25 °C for 1 h. Sequencing-grade modified trypsin was used for in-solution digestion with trypsin: protein at 1:30 (w/w).
The tryptic peptides were then subjected to liquid chromatography–mass spectrometry (LC-MS/MS) for quantitative analysis. The peptides were loaded onto a 2.1 mm × 150 mm reverse-phase column (Zorbax SB-C18, Agilent) and eluted with gradient mobile phase solution into an Agilent 6520b Q-TOF mass spectrometer37 (link). Data acquisition was performed in the auto MS-MS mode of MassHunter software (version B03.01, Agilent). The spectra were processed using SpectrumMill MS Proteomics Workbench (version A.03.03, Agilent) and searched against the NCBI green algae protein database (retrieved on April 21, 2013). The precursor mass tolerance was set at ± 20 ppm. All protein identifications with protein score ≥20 and peptide SPI ≥60% were considered as positive results. Finally, gene ontology (GO) analysis was conducted for protein function analysis.

Wang H., Lin A., Gu W., Huan L., Gao S, & Wang G. (2016). The sporulation of the green alga Ulva prolifera is controlled by changes in photosynthetic electron transport chain. Scientific Reports, 6, 24923.

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MS/MS Peptide Identification Pipeline

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MS/MS spectra from triplicate runs were exported and searched against an MS/MS database to identify proteins and peptides on the Spectrum Mill MS Proteomics Workbench (Agilent Technologies). The search parameters were as follows: precursor mass tolerance, 20 ppm; product ion mass tolerance, 50 ppm; maximum ambiguous precursor charge, 3; 2 missed cleavages allowed; fully digested peptide by trypsin; fixed modification of carbamidomethyl cysteine; variable modifications of oxidized methionine; and N-terminal carbamylation. Autovalidation of MS/MS spectra was processed with default value. FDR threshold was 1.2.

Mun S., Lee J., Lim M.K., Lee Y.R., Ihm C., Lee S.H, & Kang H.G. (2018). Development of a Novel Diagnostic Biomarker Set for Rheumatoid Arthritis Using a Proteomics Approach. BioMed Research International, 2018, 7490723.

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Shannon Entropy-Based Mass Spectrometry Protocol

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Shannon Entropy is a measure of the information content of a message or data set.

H = - \sum_{i} P (x_{i}) l o g_{b} (P (x_{i}))

H is the Shannon Entropy, so named because it resembles the Boltzman equation for thermodynamic entropy. P can be any data. In this case is the intensity at an m/z ratio of x. The equation sums the product of the P(x) times the log(P(x)) over all i points in the mass spectrum. The base of logarithm can be any number, in this case b=10. In data not shown, there was very little discriminatory gain to utilizing different bases. For each mass spectrum analyzed here, the ion peak lists were generated using the Data Extractor function of Spectrum Mill MS Proteomics Workbench (Agilent) and binned into vectors of length i, with a cumulative intensity in each bin. The intensities for each vector were normalized prior to calculating H.
After sorting the values of H for the spectra with the lowest values, the second step of the algorithm was to check for the ion pattern typical of the Amadori products, namely that the maximum abundance ion had a neutral loss of a water and a loss of HCHO. All of the ions in the top 10% of intensities were checked for two corresponding mass peaks at the 2+, 3+, or 4+ states for 3H₂O and HCHO, with a mass tolerance of 2 Da. Both the Shannon entropy and ion pattern based filtering steps are evaluated below.

Johnson K.L., Williams J.G., Maleki S.J., Hurlburt B.K., London R.E, & Mueller G.A. (2016). Enhanced approaches for identifying Amadori products: application to peanut allergens. Journal of agricultural and food chemistry, 64(6), 1406-1413.

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