Scaffold program

Manufactured by Proteome Software

Sourced in United Kingdom, United States

Scaffold is a software program designed for the analysis and visualization of proteomics data. It provides a comprehensive solution for processing, interpreting, and managing mass spectrometry-based proteomic data. The core function of Scaffold is to facilitate the identification and quantification of proteins from complex biological samples.

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Lab products found in correlation

Mascot search engine, by Matrix Science (4 mentions) Mascot, by Matrix Science (3 mentions) Mascot search engine program, by Matrix Science (3 mentions) Elastase, by Promega (3 mentions) Q exactive plus orbitrap, by Thermo Fisher Scientific (3 mentions) Trypsin, by Promega (3 mentions) Imagemaster 2d platinum software, by GE Healthcare (1 mentions) Mascot program, by Matrix Science (1 mentions) Ultraflex 3 maldi tof tof mass spectrometer, by Bruker (1 mentions) Flag peptide, by Merck Group (1 mentions) Sinapinic acid, by Merck Group (1 mentions) Ultraflex 3 tof tof instrument, by Bruker (1 mentions) Sonifier 250, by Emerson (1 mentions)

13 protocols using scaffold program

Bovidae Protein Identification via MALDI-TOF

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Peptide masses of the samples were obtained using an Ultraflex III MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) and then screened against the Bovidae database deposited in UniProt using the MASCOT program (Matrix Science, London, UK) and a MASCOT Peptide Mass Fingerprinting database search. An accuracy of 0.5 Da was used in the search criteria. Trypsin was set as an enzyme with one allowed miscleavage. The fixed modification and variable modification factors used were carbamidomethyl and oxidation, respectively. The numbers of peptide matches, sequence coverage, molecular weight (MW), and isoelectric point (pI) were used to evaluate the database search results. The Scaffold program (Proteome Software, Portland, OR) was used to validate the proteins identified by the MASCOT program, with the identity for both proteins and peptides equal to 100%.
Protein abundance was quantified by spot densitometry analysis using ImageMaster 2D Platinum Software (GE Health Care, Little Chalfont-UK). The software used powerful algorithms for efficient spot detection, accurate spot quantification, and an accurate statistical comparison of spot density across gels.

Carvalho E.B., Gionbelli M.P., Rodrigues R.T., Bonilha S.F., Newbold C.J., Guimarães S.E., Silva W., Verardo L.L., Silva F.F., Detmann E, & Duarte M.S. (2019). Differentially expressed mRNAs, proteins and miRNAs associated to energy metabolism in skeletal muscle of beef cattle identified for low and high residual feed intake. BMC Genomics, 20, 501.

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Flag-Affinity Purification and Mass Spectrometry

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3×Flag-ATX-3 transiently expressed in HEK293T cells was immunoprecipitated with anti-Flag M2 affinity gel and eluted with 3×Flag peptide (Sigma). Eluted proteins were identified with a gel-based liquid chromatography-tandem mass spectrometry (Gel-LC-MS/MS) approach and ion trap mass spectrometry (LTQ; Thermo Electron). A Mascot database search and the Scaffold program (Proteome Software) were used to visualize and validate results.

Liu H., Li X., Ning G., Zhu S., Ma X., Liu X., Liu C., Huang M., Schmitt I., Wüllner U., Niu Y., Guo C., Wang Q, & Tang T.S. (2016). The Machado–Joseph Disease Deubiquitinase Ataxin-3 Regulates the Stability and Apoptotic Function of p53. PLoS Biology, 14(11), e2000733.

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Shotgun Proteomics of High and Low Feed Efficiency Broilers

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All procedures for animal care complied with the University of Arkansas Institutional Animal Care and Use Committee (IACUC): Protocol #14012. A complete description of the procedures for this study is provided in Kong et al. (2016). Briefly, following protein extraction from breast muscle tissue (∼100 mg) obtained from PedM broilers exhibiting high and low FE phenotypes (n = 4 per group), proteins were subjected to shotgun proteomic analysis by in-gel trypsin digestion and tandem mass spectrometry (MS/MS) at the University of Arkansas for Medical Sciences (UAMS) Proteomics Core Lab (Little Rock, AR). Raw spectrometric data was analyzed by the Mascot (Matrix Science, Boston, MA) database search engine along with the UniProtKB (http://www.uniprot.org/help/uniprotkb) database, and compiled using the Scaffold program (Proteome Software, Portland OR). Normalization of data based on total spectral counts for each individual sample revealed no differences in actin, myosin, or tubulin expression that were used as house-keeping proteins in this study. A search for “CKMT” in the excel file of the normalized proteomic dataset revealed two CKMT protein isoforms (CKMT1A and CKMT2) that had been identified and given separate accession numbers. However, both isoforms but had not been assigned a gene name and therefore were not previously reported by Kong et al. (2016).

Bottje W.G., Lassiter K., Dridi S., Hudson N, & Kong B.W. (2017). Enhanced expression of proteins involved in energy production and transfer in breast muscle of pedigree male broilers exhibiting high feed efficiency. Poultry Science, 96(7), 2454-2458.

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Shotgun Proteomics Analysis via Mass Spectrometry

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Extracted individual proteins were subjected to shotgun proteomics analysis by in-gel trypsin digestion and tandem mass spectrometry (MS/MS) at the University of Arkansas Medical Science (UAMS) Proteomics Core Lab (Little Rock, AR). In total, 25 gel slices per sample were analyzed. Raw mass spectrometric data were analyzed by database searching using the Mascot (Matrix Science, Boston, MA) search engine and the UniProtKB (http://www.uniprot.org/help/uniprotkb) database. Search results were compiled using Scaffold program (Proteome Software, Portland, OR).

Kong B.W., Lassiter K., Piekarski-Welsher A., Dridi S., Reverter-Gomez A., Hudson N.J, & Bottje W.G. (2016). Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria. PLoS ONE, 11(5), e0155679.

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Protein Identification via LC-MS/MS

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LC–MS/MS data were searched against an in-house protein sequence database containing Swiss-Prot and the protein constructs specific to the experiment, using the Mascot search engine program (Matrix Science, version 2.4). Database search parameters were set with a precursor tolerance of 5 p.p.m. and a fragment ion mass tolerance of 0.8 Da. Variable modifications for oxidized methionine, carbamidomethyl cysteine, pyroglutamic acid, and deamination of glutamine/asparagine were included. MS/MS data were validated using the Scaffold program (version 5, Proteome Software Inc.).

Tang S., Beattie A.T., Kafkova L., Petris G., Huguenin-Dezot N., Fiedler M., Freeman M, & Chin J.W. (2022). Mechanism-based traps enable protease and hydrolase substrate discovery. Nature, 602(7898), 701-707.

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Mass Spectrometry-Based Peptide Analysis

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Mass spectrometry was performed by the LMB Mass Spectrometry Facility. Briefly, peptides from in situ trypsin digestion were extracted in 2% formic acid/2% acetonitrile mix. Digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC and C18 Acclaim PepMap100 nanoViper (Thermo Scientific Dionex). LC-MS/MS data were searched against a protein database (UniProt KB) with the Mascot search engine program (Matrix Science). MS/MS data were validated using the Scaffold program (Proteome Software). MALDI-TOF mass spectrometric measurements were carried out in positive ion mode on an Ultraflex III TOF-TOF instrument (Bruker Daltonik), using sinapinic acid (Sigma) as matrix.

Gammons M.V., Renko M., Flack J.E., Mieszczanek J, & Bienz M. (2020). Feedback control of Wnt signaling based on ultrastable histidine cluster co-aggregation between Naked/NKD and Axin. eLife, 9, e59879.

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Shotgun Proteomic Analysis of Broiler Plasma

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Plasma collected from Con and WB broilers at 4 and 8 wks of age (32 plasma samples in total) were subjected to shotgun proteomics analysis by trypsin digestion and tandem mass spectrometry (MS/MS) at the University of Arkansas Medical Science (UAMS) Proteomics Core Facility (Little Rock, AR). Raw mass spectrometric data were analyzed by database searching using the Mascot (Matrix Science, Boston, MA) search engine and the UniProtKB (http://www.uniprot.org/help/uniprotkb) database. Search results were compiled using Scaffold program (Proteome Software, Portland, OR). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019 (link)) partner repository with the dataset identifier PXD026896 and 10.6019/PXD026896. Normalization and statistical analyses are described in the “Statistical Analyses” section.

Kong B., Khatri B., Kang S., Shouse S., Kadhim H., Kidd M J.r., Lassiter K., Hiltz J., Mallmann B., Orlowski S., Anthony N., Bottje W., Kuenzel W, & Owens C. (2021). Blood Plasma Biomarkers for Woody Breast Disease in Commercial Broilers. Frontiers in Physiology, 12, 712694.

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Affinity Purification of UBR5-Associated Proteins

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For affinity purification of UBR5-associated proteins, 20x 175 cm² flasks of HEK293T cells transfected with UBR5 or control baits were used for each experiment. Cells were lysed in 40 mL lysis buffer (20 mM Tris-HCl pH 7.4, 10% glycerol, 100 mM NaCl, 5 mM NaF, 2 mM Na₃PO₄, 0.2% Triton X-100, protease inhibitor cocktail), and sonicated 10x 10 s at 40% intensity with a Branson 250 Sonifier. Cell lysates were clarified by centrifugation (21,000x g, 30 min, 4°C) and incubated (while rotating) for 2 hr with Flag affinity gel at 4°C. Immunoprecipitates were washed 5x with lysis buffer, and subsequently eluted with lysis buffer supplemented with 250 μg mL⁻¹ 3xFlag-Peptide. Eluates were boiled in LDS sample buffer and resolved on 4%–12% Bis-Tris SDS-polyacrylamide gels. These were stained with Imperial Protein Stain, and gel lanes cut into 1-2 mm slices for in situ trypsin digestion. Resulting peptides were extracted in 2% formic acid / 2% acetonitrile mix. Digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC and C18 Acclaim PepMap100 nanoViper (Thermo Scientific Dionex). LC-MS/MS data were searched against a protein database (UniProt KB) with the Mascot search engine program (Matrix Science). MS/MS data were validated using the Scaffold program (Proteome Software).

Flack J.E., Mieszczanek J., Novcic N, & Bienz M. (2017). Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5. Molecular Cell, 67(2), 181-193.e5.

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Shotgun Proteomics Analysis of Proteins

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Individual extracted proteins were used in shotgun proteomics analysis with in-gel trypsin digestion followed by tandem mass spectrometry (MS/MS) conducted at the University of Arkansas for Medical Science proteomics core lab (UAMS, Little Rock, AR, USA) [11 (link)]. Raw data were analyzed by database searching using Masco (Matrix Science, Boston, MA, USA) and UniProtKB database (http://www.uniprot.org/help/uniprotkb) with the result compiled using the Scaffold Program (Proteome Software, Portland, OR, USA).
In Kuttappan et al. [2 (link)], digested sample homogenates were acidified with 1% formic acid, and purified by reversed phase chromatography using C18 affinity media (Omix-Agilent, Santa Clara, CA, USA). Each sample was subjected to three replicate analyses for LC-MS/MS using a hybrid-OrbitrapXL mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) according to Voruganti et al. [14 (link)]. Mass spectrometry analysis was conducted in the DNA/Protein Resource Facility (Oklahoma State University, Stillwater, OK, USA).

Bottje W.G., Lassiter K.R., Kuttappan V.A., Hudson N.J., Owens C.M., Abasht B., Dridi S, & Kong B.C. (2021). Upstream Regulator Analysis of Wooden Breast Myopathy Proteomics in Commercial Broilers and Comparison to Feed Efficiency Proteomics in Pedigree Male Broilers. Foods, 10(1), 104.

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Proteomic analysis of purified proteins

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Purified proteins were prepared for mass spectrometric analysis by in solution enzymatic digestion, without prior reduction and alkylation. Protein samples were digested with trypsin or elastase (Promega), both at an enzyme to protein ratio of 1:20. The resulting peptides were analysed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC (ThermoScientific Dionex) to deliver a flow of approximately 300 nl/min. A C18 Acclaim PepMap100 5 µm, 100 µm x 20 mm nanoViper (ThermoScientific Dionex), trapped the peptides prior to separation on a C18 Acclaim PepMap100 3 µm, 75 µm x 250 mm nanoViper (ThermoScientific Dionex, San Jose, USA). Peptides were eluted with a 90 min gradient of acetonitrile (2% to 50%). The analytical column outlet was directly interfaced via a nano-flow electrospray ionization source, with a hybrid quadrupole orbitrap mass spectrometer (Q-Exactive Plus Orbitrap, ThermoScientific). LC-MS/MS data were then searched against an in house LMB database using the Mascot search engine (Matrix Science) 72 (link), and the peptide identifications validated using the Scaffold program (Proteome Software Inc.) 73 (link). All data were additionally interrogated manually.

Alfieri C., Chang L., Zhang Z., Yang J., Maslen S., Skehel M, & Barford D. (2016). Molecular basis of APC/C regulation by the spindle assembly checkpoint. Nature, 536(7617), 431-436.

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