The reproducibility of proteome changes in the CRVO model was verified by comparing CRVO (n = 5) vs. laser control (n = 5) with tandem mass tag (TMT)-based mass spectrometry in a separate analytical run. Eyes from eight animals were used to compare the protein profile of CRVO + aflibercept (n = 8) vs. CRVO + NaCl (n = 8) with proteomic analysis by tandem mass tag (TMT)-based mass spectrometry.
Isobaric labeling was performed with a 10 plex TMT kit from Thermo Scientific (Waltham, MA, USA). Sample preparation for TMT-based mass spectrometry was performed as previously described [14 (link),16 (link)] with some modifications. For the experiment consisting of 16 samples, a standard was prepared by mixing equal amounts from each sample. Two groups of 10 samples, 8 experimental samples together with 2 standards, were labelled with the 10 plex kit. The standards were used for normalization of data. TMT labeling and high pH reversed phase peptide fractionation were performed as described in a previous article [18 (link)]. Then, 1 µg of fractions 2–8 was analyzed.
Isobaric labeling was performed with a 10 plex TMT kit from Thermo Scientific (Waltham, MA, USA). Sample preparation for TMT-based mass spectrometry was performed as previously described [14 (link),16 (link)] with some modifications. For the experiment consisting of 16 samples, a standard was prepared by mixing equal amounts from each sample. Two groups of 10 samples, 8 experimental samples together with 2 standards, were labelled with the 10 plex kit. The standards were used for normalization of data. TMT labeling and high pH reversed phase peptide fractionation were performed as described in a previous article [18 (link)]. Then, 1 µg of fractions 2–8 was analyzed.
Free full text: Click here
