Spectrum mill

Manufactured by Agilent Technologies

Sourced in United States

The Spectrum Mill is a laboratory instrument that performs spectral analysis. It is designed to measure and analyze the spectrum of electromagnetic radiation emitted or absorbed by a sample. The core function of the Spectrum Mill is to provide accurate and reproducible spectral data to support various scientific and analytical applications.

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29 protocols using spectrum mill

Proteomics Analysis of Tomato Proteins

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The band from the SDS-PAGE was excised, cut into small pieces and in-gel digested: trypsin was chosen as proteolytic enzyme. Protein digestion was performed according to the manufacturer’s instructions. The tryptic digest was analyzed by LC-nano-ESI-ion trap analysis (LC-MS/MS) (LC/MCD-Trap-XCT-Ultra, Agilent-Technologies, Palo Alto, CA). Peptide separation was performed using a Zorbax 300SB reverse phase C18 column (150 mm × 0.075 mm, 3.5 µm). The following conditions were used for the analytical separation: 5–70% acetonitrile gradient in 0.1% formic acid over 55 min, with a flow rate of 0.3 µL/min. Spectra acquisition was performed in Data-dependent scan modality and analyzed using Mascot Search (http://www.matrixscience.com/) and Spectrum Mill (Agilent Technologies, Palo Alto, CA, USA) software. The protein search was performed against a customized database (UniProt DB, available online) containing approximately 36,880 entries referred to Lycopersicon esculentum species.

Loi M., De Leonardis S., Mulè G., Logrieco A.F, & Paciolla C. (2020). A Novel and Potentially Multifaceted Dehydroascorbate Reductase Increasing the Antioxidant Systems is Induced by Beauvericin in Tomato. Antioxidants, 9(5), 435.

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LC-MS Differential Proteomic Analysis of Rat Serum

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Identified spectra were used to populate an accurate mass and time (AMRT) database. Unfractionated digested rat serum samples were analyzed by LCMS for differential analysis. Peptides which passed differential filters (ANOVA and fold change, see Statistics section) were identified in one of two ways: differential peptides were used to search the AMRT database. The remaining peptides without database hits were then targeted for by LCMS/MS and the resulting spectra were searched against the SwissProt Rattus Norvegicus database in Spectrum Mill (Agilent, Santa Clara, CA) allowing up to 2 missed tryptic cleavages with variable carbamidomethyl (C), deamidated (N), oxidation (M), N-term pryroglutamic acid (Q), and phosphorylated (STY) modifications. Data was extracted and aligned for mass and time using a recursive strategy performed in Profinder software (Agilent) and Mass Profiler Professional software (Agilent). Peptides were annotated using ID Browser software (Agilent) by matching mass and retention time from aligned experimental data to peptide entries in the AMRT library generated previously from MS/MS data. The annotated peptide list was filtered to peptides that were found in at least 4 of 6 samples and in at least 1 of 3 conditions. Peptides were rolled up into protein abundances using sort and subtotal functions within Microsoft Excel.

Thompson L.C., Shannahan J.H., Perez C.M., Haykal-Coates N., King C., Hazari M.S., Brown J.M, & Farraj A.K. (2019). Early Proteome Shift and Serum Bioactivity Precede Diesel Exhaust-induced Impairment of Cardiovascular Recovery in Spontaneously Hypertensive Rats. Scientific Reports, 9, 6885.

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Proteomic Analysis of Recombinant Ara h 1

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Peak lists were generated from the data obtained from the nanoLC-ESI-MS/MS analysis using the Data Extractor feature of the Spectrum Mill software from Agilent. The Data Extractor settings included limiting the data search to deconvoluted ions observed between 400 and 6000 Da and a retention time between 10 minutes and 50 minutes. Moreover, of the remaining MS/MS spectra, only spectra that contained sequence tag information greater than or equal to 1 residue were submitted for database searching. The resulting extracted data were then searched iteratively against the sequence of the recombinant Ara h 1 (gi:347447590) using the MS/MS Search function in the Spectrum Mill software. Search settings included a trypsin specificity with two missed cleavages allowed, a precursor ion mass tolerance of 2 Da, a product ion mass tolerance of 0.7 Da, variable methionine oxidation, and a minimum matched spectral intensity of 70%. Putative matches were manually validated and the remaining unmatched spectra were then searched again against the rAra h 1 sequence using similar settings but allowing for lysine modification by either carboxymethyl-, carboxyethyl, or Amadori products.

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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Proteomic Analysis of H. pylori Infection in RAW 264.7 Macrophages

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RAW 264.7 macrophages were infected with H. pylori PMSS1 for 24 h and lysed in a solution containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, and 2 mM EDTA. After centrifugation at 16,000 × g for 15 min, the protein concentration in the supernatants was determined by a BCA assay. Protein samples were precipitated with ice-cold acetone overnight at −20°C, washed, and digested with sequencing-grade trypsin overnight. Next, proteomic analysis was performed using iTRAQ technology. Upon gradient elution, peptides were introduced via nanospray ionization into a Q Exactive HF mass spectrometer (Thermo Scientific). Peptide/protein identifications and quantitative analysis were performed using Spectrum Mill (Agilent). Tandem mass spectrometry (MS/MS) spectra were searched against a subset of the UniProt KB protein database containing Mus musculus protein sequences. Autovalidation procedures in Spectrum Mill were used to filter the data to <1% false discovery rates at the protein and peptide levels. The median log₂ iTRAQ protein ratios were calculated over all peptides identified for each protein and fit using least-squares regression, and P values were corrected for multiple comparisons by the Benjamini-Hochberg method (64 ).

Gobert A.P., Latour Y.L., Asim M., Finley J.L., Verriere T.G., Barry D.P., Milne G.L., Luis P.B., Schneider C., Rivera E.S., Lindsey-Rose K., Schey K.L., Delgado A.G., Sierra J.C., Piazuelo M.B, & Wilson K.T. (2019). Bacterial Pathogens Hijack the Innate Immune Response by Activation of the Reverse Transsulfuration Pathway. mBio, 10(5), e02174-19.

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Proteomic Analysis of Malvales Plants

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Data processing and protein identification were performed using Spectrum Mill (Ver. B.06.00.201, Agilent Technologies, USA) against an organism-specific database (NCBI taxa: Malvales; as of October 2016). Carbamidomethylation was selected as the fixed modification parameter and trypsin as the digestion enzyme with the maximum number of missed cleavages equal to 2. The MS searches were auto-validated at a false discovery rate (FDR) of 1.2%, followed by the generation of protein–protein comparison files and data export to Mass Profiler Professional (MPP v 14.9.1, Agilent Technologies, USA) for further analysis. The data files were then processed by principle component analysis (PCA) using the MPP software.

Hishamuddin M.S., Lee S.Y., Isa N.M., Lamasudin D.U., Zainal Abidin S.A, & Mohamed R. (2019). Time-based LC-MS/MS analysis provides insights into early responses to mechanical wounding, a major trigger to agarwood formation in Aquilaria malaccensis Lam. RSC Advances, 9(32), 18383-18393.

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Proteomic Identification and Quantification

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All .raw files were searched using Spectrum Mill (Agilent Technologies). MS2 spectra were searched against the Uniprot Mouse database (10/17/2014, 41,309 mouse entries, 150 common laboratory contaminants), with a mass tolerance of 20 ppm for both the precursor and product ions. The enzyme specificity was set for LysC/Trypsin and allowing up to three missed cleavages. The fixed modification was carbamidomethylation at cysteine. TMT labeling was required at lysine, but peptide N–termini were allowed to be either labeled or unlabeled. Allowed variable modifications for whole proteome datasets were acetylation of protein N–termini, oxidized methionine, deamidation of asparagine, and pyroglutamic acid at peptide N–terminal glutamine, with a precursor MH⁺ shift range of 18 to 64 Da. The false discovery rate was determined to be less than 1%. For proteome interpretation, protein identifications were discarded if the protein was only observed by a single peptide. For reporting number of proteins identified protein subgroups, protein gene products (proteoforms) from the same gene, were expanded to account for the observation of different proteoforms. For statistical tests protein subgroups were collapsed to the proteoform with the most evidence.

Katrancha S.M., Shaw J.E., Zhao A.Y., Myers S.A., Cocco A.R., Jeng A.T., Zhu M., Pittenger C., Greer C.A., Carr S.A., Xiao X, & Koleske A.J. (2019). Trio Haploinsufficiency Causes Neurodevelopmental Disease–Associated Deficits. Cell reports, 26(10), 2805-2817.e9.

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Quantitative Phosphoproteomics of Peptide Signaling

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Arabidopsis thaliana T87 suspension-cultured cells and maize suspension-cultured endosperm cells (var. Black Mexican Sweet) were treated with either water or 100 nM AtPep1 and ZmPep3, respectively. Cells were harvested after 10 min into liquid nitrogen. Protein extracted from these samples was subjected to a tryptic digest, labeled with iTRAQ mass tags, mixed and analyzed. Phosphopeptides were enriched using CeO₂ affinity capture and analyzed separately. Peptides were separated by nano-LC using salt gradients on a three-phase capillary column with an LTQ Velos linear ion trap tandem MS in positive ion mode and data-dependent acquisitions^{67 (link)}. Peptides were separated into three mass classes prior to scanning and each MS scan was followed by five MS/MS scans of the most intense parent ions. Data was extracted and searched using Spectrum Mill (Agilent). Peptide abundance and phosphorylation levels were quantified by spectral counting with counts for each protein representing the total number of peptides that matches to that protein.

Dressano K., Weckwerth P.R., Poretsky E., Takahashi Y., Villarreal C., Shen Z., Schroeder J.I., Briggs S.P, & Huffaker A. (2020). Dynamic regulation of Pep-induced immunity through post-translational control of defense transcript splicing. Nature plants, 6(8), 1008-1019.

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Proteomic Analysis of Human Proteins

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MS analysis was performed as described previously.^{52 (link)} Briefly, bound proteins were digested by trypsin (Roche) directly on beads for the MS analysis. Digested peptides were separated by online 2D-nanoLC and detected by LTQ linear ion-trap mass spectrometers. Each sample took 22.5 h to analyze, and about 200,000 MS/MS spectra were collected for each run. Raw data were extracted and searched by using Spectrum Mill (Agilent, v3.03) database search software against the NCBI ref seq database limited to human taxonomy (version 44).

Hasan M.K., Widhopf GF I.I., Zhang S., Lam S.M., Shen Z., Briggs S.P., Parker B.A, & Kipps T.J. (2019). Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis. NPJ Breast Cancer, 5, 35.

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Q-TOF-MS Database Search Protocol

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The available data obtained from the samples by the Q-TOF-MS were searched against the animal species sub-directory of the SwissProt.Holothuridae.January2017.1047.fasta database (UniProt, EBI, UK) using the SpectrumMill search engine (SpectrumMill Rev.B.04.00.127, Agilent Technologies). Basic parameters were set at a precursor mass tolerance of 10 ppm, a product mass tolerance of 50 ppm and a ‘no-enzyme’ constraint. Peptides with score above 6 and a percentage of scored peak intensity higher than 50% were considered a match.

Auwal S.M., Zainal Abidin N., Zarei M., Tan C.P, & Saari N. (2019). Identification, structure-activity relationship and in silico molecular docking analyses of five novel angiotensin I-converting enzyme (ACE)-inhibitory peptides from stone fish (Actinopyga lecanora) hydrolysates. PLoS ONE, 14(5), e0197644.

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Comprehensive Proteomics Data Processing

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For heart and liver, raw MS/MS data samples were processed by a Spectrum Mill (v.7.09.215) (Agilent Technologies). For the remaining tissues, sample processing was implemented by an in-house cloud-based proteomics pipeline executed in the Google Cloud Platform^{5 (link)}. In all tissues, MS2 spectra were processed and searched against the rat RefSeq protein database (downloaded November 2018). Log₂ TMT ratios to the common reference were used as quantitative values for all proteins. Principal component analysis and median protein abundance across samples were used to find sample outliers. Proteomics features that were not fully quantified in at least two plexes within a tissue and non-rat contaminants were removed. Median-centering and mean absolute deviation scaling of Log₂ TMT ratios were done for sample normalization. Plex batch effects were removed using limma::removeBatchEffect function in R (v 3.48.0).

Smith G.R., Zhao B., Lindholm M.E., Raja A., Viggars M.R., Pincas H., Gay N.R., Sun Y., Ge Y., Nair V.D., Sanford J.A., Amper M.A., Vasoya M., Smith K.S., Montgomery S.B., Zaslavsky E., Bodine S.C., Esser K.A., Walsh M.J., Snyder M, & Sealfon S.C. (2023). Multi-omic identification of key transcriptional regulatory programs during endurance exercise training. bioRxiv.

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