| Sandhya Visweswariah | GUCY2C project | 23 samples | Global proteomes MS3 quant.
=========TMT18plexA: A1,A2.........A18 & TMT13plexB: B1,B2,......B13========
==============#Sample Number_Sample Description_Mouse Number (IISc)_group_TMT18plex A_TMT13plex B============
#1_Azoxymethane + 1DSS_WT 33_WT_TMT18plex A1_TMT13plex B1
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#2_Azoxymethane + 1DSS_WT 35_WT_TMT18plex A2_
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#13_Only Azoxymethane_WT 36_WT_TMT18plex A3_TMT13plex B2
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#14_Only Azoxymethane_WT 38_WT_TMT18plex A4_
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#18_Untreated colonic mucosa_WT 44_WT_TMT18plex A5_TMT13plex B3
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#19_Untreated colonic mucosa_WT 45_WT_TMT18plex A6_
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#4_Azoxymethane + 1DSS_S8 34_S8_TMT18plex A7_TMT13plex B4
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#5_Azoxymethane + 1DSS_S8 30_S8_TMT18plex A8_
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#15_Only Azoxymethane_S8 36_S8_TMT18plex A9_TMT13plex B5
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#16_Only Azoxymethane_S8 37_S8_TMT18plex A10_
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#21_Untreated colonic mucosa_S8 44_S8_TMT18plex A11_TMT13plex B6
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#22_Untreated colonic mucosa_S8 45_S8_TMT18plex A12_
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#7_Azoxymethane + 1DSS_KO 26_KO_TMT18plex A13_TMT13plex B7
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#8_Azoxymethane + 1DSS_KO 27_KO_TMT18plex A14_
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#9_Azoxymethane + 1DSS_KO 28_KO_TMT18plex A15_
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#10_Azoxymethane + 1DSS_HET 8_HET_TMT18plex A16_TMT13plex B8
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#11_Azoxymethane + 1DSS_HET 1_HET_TMT18plex A17_
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#12_Azoxymethane + 1DSS_HET 17_HET_TMT18plex A18_
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#3_Azoxymethane + 1DSS_WT 27_WT__TMT13plex B9
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#20_Untreated colonic mucosa_WT 46_WT__TMT13plex B10
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#6_Azoxymethane + 1DSS_S8 29_S8__TMT13plex B11
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#17_Only Azoxymethane_S8 38_S8__TMT13plex B12
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#23_Untreated colonic mucosa_S8 46_S8__TMT13plex B13
====================================================================
Key: WT, wild type mouse; S8, S839I knock in mouse; KO, Gucy2c KO mouse; HET, S839I/wild type heterozygous mouse |
| They sent us 60ug of trypsin-digested peptides as dried samples. I resuspended the samples in 52.2ul of TEAB to get 1.15 ug/ul of peptides. Samples separated into two TMTpro sets. Set1 - 18plex and set 2 - 13plex.
Then 13ul (15ug peptides) of peptide solution was taken and labeled using 5ul TMTpro reagent. Upon a premix run, it was clear that the digestion of peptides was not optimal (82% 0 missed cleavages).
Then I redigested the remaining 39.2ul peptide samples (52.2 - 13ul) by adding 5ul of Trypsin per sample. To perform proteomics, 13ul (13.2ug peptides) was taken and labeled with 5ul TMTpro reagent.
First, a premix was run to assess trypsin digestion, in both TMT sets, which was found to be efficient (>97% 0 missed cleavage). The final concentration is 1.02 ug/ul (45ug in 44.2ul).
Samples were separated into 24 fractions. Set1 with 18 samples was resuspended 99ul (0.1ug/ul peptides). Set 2 with 13 samples was resuspended in 71.5ul (0.1ug/ul).
Sample left: Samples can still be used for MS runs (inject 10ul for 1ug peptide per fraction) |