Quantitative Mass Spectrometry Analysis

Purified proteins bound to streptavidin beads were reduced, alkylated, and digested by sequential addition of Lys-C and trypsin proteases^{33 (link), 34 (link)}. The peptide mixture was desalted using C₁₈ tips and fractionated online using a 75 μM inner diameter fritted fused silica capillary column with a 5 μM pulled electrospray tip, and packed in house with 15 cm of Luna C₁₈ 3 μM reversed-phase particles. The gradient was delivered by an easy-nLC 1000 ultrahigh-pressure liquid chromatography (UHPLC) system (Thermo Scientific). Tandem mass spectrometry (MS/MS) spectra were collected on a Q-Exactive mass spectrometer (Thermo Scientific). Data analysis was performed using the ProLuCID and DTASelect2 implemented in the Integrated Proteomics pipeline IP2 (Integrated Proteomics Applications, Inc., San Diego, CA). Protein and peptide identifications were filtered using DTASelect and required a minimum of two unique peptides per protein and a peptide-level false-positive rate of less than 5%, as estimated by a decoy database strategy. Normalized spectral abundance factor (NSAF) values were calculated as described^{33 (link)} and the data were analyzed using the Pathway Studio software^{34 (link), 35 (link)}.

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Mota C.M., Chen A.L., Wang K., Nadipuram S., Vashisht A.A., Wohlschlegel J.A., Mineo T.W, & Bradley P.J. (2017). RETRACTED ARTICLE: New molecular tools in Neospora caninum for studying apicomplexan parasite proteins. Scientific Reports, 7, 3768.

Publication 2017

easy-nLC 1000 ultrahigh-pressure liquid chromatography (UHPLC) system Q-Exactive mass spectrometer

Capillary Liquid chromatography Peptide Pressure Protein Silica Spectrometry Streptavidin Tandem mass spectra Trypsin

Corresponding Organization : University of California, Los Angeles

Top 5 similar protocols

Variable analysis

independent variables

Purified proteins bound to streptavidin beads
Sequential addition of Lys-C and trypsin proteases

dependent variables

Peptide mixture
Tandem mass spectrometry (MS/MS) spectra
Protein and peptide identifications
Normalized spectral abundance factor (NSAF) values

control variables

Reduction and alkylation of purified proteins
Desalting of peptide mixture using C18 tips
Fractionation of peptide mixture using a 75 μM inner diameter fritted fused silica capillary column packed with 15 cm of Luna C18 3 μM reversed-phase particles
Gradient delivery by an easy-nLC 1000 ultrahigh-pressure liquid chromatography (UHPLC) system
Data analysis using ProLuCID, DTASelect2, and Integrated Proteomics pipeline IP2
Protein and peptide identification filtering using DTASelect with a minimum of two unique peptides per protein and a peptide-level false-positive rate of less than 5%
Data analysis using Pathway Studio software

positive controls

Not specified

negative controls

Not specified

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