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Scaffold v4.4.1.1

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Scaffold (v4.4.1.1) is a software application used for the analysis and visualization of mass spectrometry proteomics data. It provides a platform for the identification and quantification of proteins from complex biological samples.

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Lab products found in correlation

Q exactive quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions) Water xbridge column c18 3.5um, by Agilent Technologies (2 mentions) Easy nlc, by Thermo Fisher Scientific (2 mentions) Mascot, by Matrix Science (2 mentions) Immobilon p, by Merck Group (1 mentions) Irdye 800, by LI COR (1 mentions) Fastprep cell homogenizer, by MP Biomedicals (1 mentions) Irdye 680lt, by LI COR (1 mentions) Anti gfp, by Roche (1 mentions)

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Scaffold 4 (92 citations) Scaffold v4.4.6 (22 citations)

3 protocols using scaffold v4.4.1.1

1

Immunoprecipitation and Mass Spectrometry Analysis

Cited 2 times
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Cell pellets were snap frozen in dry ice–ethanol baths. Lysates were prepared using a Fastprep cell homogenizer (MP Biomedicals). Immunoprecipitations were performed as previously described (Gould et al., 1991 (link)) in NP-40 buffer for native lysates. Protein samples were resolved by SDS-PAGE and transferred to PVDF membrane (Immobilon P; EMD Millipore). Anti-HA (12CA5), anti-Flag (M2; Sigma-Aldrich), anti-GFP (Roche), anti-Cdc15 serum (Roberts-Galbraith et al., 2009 (link)), or anti-PSTAIRE (Cdc2; Sigma-Aldrich) were used in immunoprecipitations and/or as primary antibodies in immunoblotting. Secondary antibodies were conjugated to IRDye680LT or IRDye800 (LI-COR Biosciences). Blotted proteins were detected via Odyssey (LI-COR Biosciences).
Purification of Efr3-TAP and subsequent identification of interacting proteins by mass spectrometry were performed as previously described (Gould et al., 2004 (link); Chen et al., 2013 (link); Elmore et al., 2014 (link)) with the following changes: a newer version of Scaffold (Scaffold v4.4.1.1; Proteome Software) was used, and the minimum peptide identification probability was changed to 95.0%.
All ANOVA statistical analyses used Tukey’s post hoc analysis.
Snider C.E., Willet A.H., Chen J.S., Arpağ G., Zanic M, & Gould K.L. (2017). Phosphoinositide-mediated ring anchoring resists perpendicular forces to promote medial cytokinesis. The Journal of Cell Biology, 216(10), 3041-3050.
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2

Peptide Quantification and Identification Workflow

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Samples were cleaned using reversed phase liquid chromatography as previously described34 (link) (high pH RP, Agilent 1200 series micro-flow pump, Water XBridge column C18 3.5um, 2.1×100mm). The samples were dried (speed vacuum) and stored at −80 °C until quantification using LavaPep’s Fluorescent Peptide and Protein Quantification Kit (Gel Company) according to manufacturer’s protocol. Equal amounts of peptides for each sample were re-suspended (2% acetonitrile, 0.1% formic acid) and injected into a nano flow liquid chromatography system (Easy nLC, Thermo Fisher) connected inline to a Q Exactive Quadrupole Orbitrap mass spectrometer. Raw MS spectra were processed by Progenesis (Nonlinear Dynamics) and Mascot (Matrix Science) as previously described35 (link), 36 (link). Technical variability was determined by the addition of a protein mix of all the samples that was injected in 10 sample intervals. Search results were entered into Scaffold (v4.4.1.1; Proteome Software, Portland, OR) to determine protein identifications (80% peptide confidence; 95% protein confidence, with minimum of 2 unique peptides per protein).
Hensley-McBain T., Berard A.R., Manuzak J.A., Miller C.J., Zevin A.S., Polacino P., Gile J., Agricola B., Cameron M., Hu S.L., Estes J.D., Reeves R.K., Smedley J., Keele B.F., Burgener A.D, & Klatt N.R. (2018). Intestinal damage precedes mucosal immune dysfunction in SIV infection. Mucosal immunology, 11(5), 1429-1440.
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3

Peptide Quantification and Identification Workflow

Cited 2 times
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Samples were cleaned using reversed phase liquid chromatography as previously described34 (link) (high pH RP, Agilent 1200 series micro-flow pump, Water XBridge column C18 3.5um, 2.1×100mm). The samples were dried (speed vacuum) and stored at −80 °C until quantification using LavaPep’s Fluorescent Peptide and Protein Quantification Kit (Gel Company) according to manufacturer’s protocol. Equal amounts of peptides for each sample were re-suspended (2% acetonitrile, 0.1% formic acid) and injected into a nano flow liquid chromatography system (Easy nLC, Thermo Fisher) connected inline to a Q Exactive Quadrupole Orbitrap mass spectrometer. Raw MS spectra were processed by Progenesis (Nonlinear Dynamics) and Mascot (Matrix Science) as previously described35 (link), 36 (link). Technical variability was determined by the addition of a protein mix of all the samples that was injected in 10 sample intervals. Search results were entered into Scaffold (v4.4.1.1; Proteome Software, Portland, OR) to determine protein identifications (80% peptide confidence; 95% protein confidence, with minimum of 2 unique peptides per protein).
Hensley-McBain T., Berard A.R., Manuzak J.A., Miller C.J., Zevin A.S., Polacino P., Gile J., Agricola B., Cameron M., Hu S.L., Estes J.D., Reeves R.K., Smedley J., Keele B.F., Burgener A.D, & Klatt N.R. (2018). Intestinal damage precedes mucosal immune dysfunction in SIV infection. Mucosal immunology, 11(5), 1429-1440.
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