Quantitative Proteomics Using iTRAQ

Protein digestion, labelling and mass spectrometry was done as described in detail previously^{28 (link)}. 30 kDa size exclusion filters (Pall Corporation) were used in sample preparation. Samples were labelled with the isobaric iTRAQ tags as shown in Fig. 1 and pooled together for the mass spectrometry analysis. Labelled samples were analysed by Nano-RPLC- TripleTOF instrumentation using Eksigent ekspert™ 425 NanoLC coupled to high speed TripleTOF™ mass spectrometer (Sciex 5600+). A capillary RP-LC column (cHiPLC® ChromXP C18-CL, 3 µm particle size, 120 Å, 75 µm i.d × 15 cm; Eksigent, Concord, Canada) was used for LC separation of peptides.

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Hongisto H., Jylhä A., Nättinen J., Rieck J., Ilmarinen T., Veréb Z., Aapola U., Beuerman R., Petrovski G., Uusitalo H, & Skottman H. (2017). Comparative proteomic analysis of human embryonic stem cell-derived and primary human retinal pigment epithelium. Scientific Reports, 7, 6016.

Publication 2017

Sciex 5600+).

Capillary Mass spectrometry Peptides Protein digestion

Corresponding Organization : Tampere University

Other organizations : University of Szeged

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Variable analysis

independent variables

Protein digestion and labelling
30 kDa size exclusion filters used in sample preparation
Isobaric iTRAQ tags used for labelling

dependent variables

Peptide separation and mass spectrometry analysis

control variables

Sample preparation method
Nano-RPLC-TripleTOF instrumentation used for mass spectrometry analysis
Capillary RP-LC column used for LC separation of peptides

controls

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Negative controls not explicitly mentioned

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