Experimental workflow

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1 Experimental workflow Pg. 3 Lysis Protein quant Protein precipitation Pg. 4 Digest Pgs. 5&6 Label Peptides Peptide quant Ratio check Search, filter & compile data Pgs Combine Samples LC-MS3 Pg. 8 Pg. 7 Fractionate 1

2 Lysis and protein quantification 400 µl Lysis Buffer was added to each sample Lysis Buffer -50 mm Tris ph 8.5-8M Urea -1% SDS -Roche protease and phosphatase inhibitors Protein Quantification using the micro-bca assay by Pierce Sample Protein concentration (mg/ml) Total amount of protein (µg) Wt Wt Inhib Inhib Wt Wt Inhib Inhib After protein quantification lysates were immediately reduced with DTT and alkylated with iodoacetimide 2

3 Protein precipitation and digestion -170 µg of protein from each sample was precipitated using methanol/chloroform (the remaining lysate is stored at -80 o C if needed) -Digestion was performed using LysC and trypsin Protease Protease:Protein Ratio LysC 1:100 Trypsin 1:200 Protein Quantification using the micro-bca assay by Pierce Sample Peptide concentration (mg/ml) Total amount of peptide (µg) Wt Wt Inhib Inhib Wt Wt Inhib Inhib ~80 µg of each sample was labeled with TMT reagent 3

4 Tandem Mass Tags for Multiplexed Proteomics Wt-1 Wt-2 Inhib-1 Inhib-2 Wt-3 Wt-4 Inhib-3 Inhib-4 Precursor ion (MS1 scan) Reporter ion quantification (MS3 scan) Peptide Sequencing (MS2 scan) m/z m/z 4

5 Normalized Intensity Peptide labelling and ratio check (small aliquot of total peptide) ~0.2 µg of each TMT-labelled sample was mixed (1 : 1 : 1 : 1 : 1 : 1 : 1 : 1) to verify labelling success Individual normalized values The normalized intensities for each sample from the ratio check were very similar. The remainder of the labeled peptides from each sample was mixed together. Based on the ratio check no corrections were made before mixing. 5

6 AU Basic ph reverse phase (brp) sample fractionation 100 Buffer A: 5% ACN, 50 mm AmBic ph 8.0 Buffer B: 90% ACN, 50 mm AmBic ph %B Fraction size 0.37 min (~300 µl) Time, min 96-well collection plate The sample was fractionated into two sets: Set 1 consists of 12 fractions, each fraction in this set is made up of the orange numbers for an entire column, e.g., 1, 25, 49, 73. Set 2 consists of 12 fractions from the black numbers for an entire column. 6

7 Normalized intensity MS analysis One complete set (12 fractions) from HPRP was analyzed on an Orbitrap Elite mass spectrometer (one complete set is stored in the -80 o C freezer if needed) Peptides were separated using a gradient of 6 to 28% acetonitrile in 0.125% formic acid over 160 minutes. Peptides were detected (MS1) and quantified (MS3) in the Orbitrap Peptides were sequenced (MS2) in the ion trap. MS1 peptide ion chromatogram from fraction #6 Time 7

8 Results 8

9 A summary of peptide and protein numbers from 12 fractions MS2 spectra were searched using the SEQUEST algorithm against a Uniprot composite database derived from the human proteome containing its reversed complement and known contaminants. Peptide spectral matches were filtered to a 1% false discovery rate (FDR) using the target-decoy strategy combined with linear discriminant analysis. The proteins from the 12 fractions were filtered to a <1% FDR Proteins were quantified only from peptides with a summed SN threshold of >=120 Total Peptides (<1%) 107,753 Total Proteins (<1%) 9,381 Total Collapsed Proteins 7,622 Quantified Proteins 7,551 FDR percentages are shown in parentheses 9

10 7,551 quantified proteins Hierarchical clustering The normalized data was hierarchically clustered by sample and proteins 100% normalized signal 0 Proteins were normalized to the summed signal across the row. The eight samples hierarchically clustered into two distinct groups. Each group contains four samples. 10

11 Database searching, filtering and normalization I have included a spreadsheet that contains two tabs: Tab1 (Summary Data) Protein annotation, gene symbol, protein description, peptides used for quantification, averaged data and standard deviations for the two groups determined by clustering, Log 2 (ratio), p-values, Benjamini-Hochberg corrected p-values, normalized signal based on total protein intensity across all samples Tab1 (Raw Data) Protein annotation, gene symbol, protein description, peptides used for quantification, raw signal, raw standard deviation (for proteins that have more than one quantified peptide), corrected signal for each sample based on channel sum correction, normalized signal based on total protein intensity across all samples ALL data is displayed please use caution with proteins identified by one peptide only! 11

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