Separated proteins were fluorescently stained with SYPRO Ruby® (Bio-Rad Laboratories, Hercules, CA, USA), and gels were visualized using a charged coupled device (CCD) camera (VersaDoc™ Imaging system 4000 MP; Bio-Rad Laboratories). 2-DE protein patterns were analyzed and quantified using software PDQuest Advanced version 8.0.1 (Bio-Rad Laboratories). Protein spots of interest were excised from the gel and, after tryptic digestion, were analyzed by mass spectrometry using ultrafleXtreme™ matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF; Bruker Daltronik GmbH, Bremen, Germany). Database search was performed in ProteinProspector MS-Fit version 5.14.4 including Swiss-Prot database version 2015.3.5 as described in previous studies.28 (link),32 (link)
2-Dimensional Gel Electrophoresis of Plasma Proteome
Separated proteins were fluorescently stained with SYPRO Ruby® (Bio-Rad Laboratories, Hercules, CA, USA), and gels were visualized using a charged coupled device (CCD) camera (VersaDoc™ Imaging system 4000 MP; Bio-Rad Laboratories). 2-DE protein patterns were analyzed and quantified using software PDQuest Advanced version 8.0.1 (Bio-Rad Laboratories). Protein spots of interest were excised from the gel and, after tryptic digestion, were analyzed by mass spectrometry using ultrafleXtreme™ matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF; Bruker Daltronik GmbH, Bremen, Germany). Database search was performed in ProteinProspector MS-Fit version 5.14.4 including Swiss-Prot database version 2015.3.5 as described in previous studies.28 (link),32 (link)
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Corresponding Organization : Linköping University
Protocol cited in 5 other protocols
Variable analysis
- Plasma sample from each subject
- Protein patterns and abundances obtained from 2-DE analysis
- Protein identities obtained from mass spectrometry analysis
- Albumin and immunoglobulin G (IgG) depletion from plasma samples
- Protein concentration measurement using 2D-Quant Kit
- Desalting of samples using PD-10 columns
- Lyophilization of samples prior to first dimension
- Resolving of lyophilized proteins in 2-DE urea sample buffer
- Application of 100 µg total protein from each subject in the first dimension
- Separation of proteins in the second dimension using Ettan™ DALTsix Electrophoresis unit
- Fluorescent staining of separated proteins with SYPRO Ruby®
- Visualization of gels using a charged coupled device (CCD) camera
- Analysis and quantification of 2-DE protein patterns using PDQuest Advanced software
- Tryptic digestion of protein spots of interest
- Mass spectrometry analysis using MALDI-TOF
- Database search in ProteinProspector MS-Fit version 5.14.4 including Swiss-Prot database version 2015.3.5
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