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ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS
Citation:
WINNIK, W. M. AND P. A. ORTIZ. ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS. Presented at 55th American Society for Mass Spectrometry Conference on Mass Spectrometry, Indianapolis, IN, June 03 - 07, 2007.
Description:
Introduction
Membrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit poor recoveries of membrane proteins, macroporous mRP-C 18 columns have been shown to resolve membrane proteins with good recoveries. The aim of this study was to develop a LC(protein)/LC(peptide)/MSMS method for hydrophobic microsomal protein fractionation for the identification of cytochromes CYP450 in mice exposed to conazole pesticides. Large on-LC-column injection volumes were used in formic acid with addition of acetonitrile and DMSO. Up to l mg of protein was injected on mRP-C18 with hydrophobic proteins retained and most hydrophilic proteins eluted in the flow¬through.
Methods
Mouse microsomal protein pellets, 1 mg, were dissolved in 1mL of 80% formic acid with (500, 1000¿1 size injections) or without (300 and 350¿1 size injections) 10% DMSO and 10% acetonitrile and injected onto a 4.6 mm x 5 cm mRP-C 18 protein column kept at 78C. Exceeding the maximum recommended sample size was acceptable because of the preferential retention of hydrophobic proteins; demonstrated by HPLC-UV and by LC/MS chromatograms of the digest. Protein HPLC gradient: 0-80% "0.1 % TFA in acetonitrile, B" in 60 min (0.75 ml/min), 98% "B" in 5 min, isocratic at 98% "B" for 10 minutes. 25 LC two-minute fractions were collected. Neutralized (pH=8) fractions were treated with RapigestTM, reduced in volume and trypsinized under standard conditions.
Preliminary results
In 25 protein fraction digests 37 cytochromes P450 were identified by LC/MSMS (using LXQ ion trap-MS with dual LC pumps and a nanospray probe) with 500¿g on-column protein sample injection in 10% DMSO, 10% acetonitrile and 80% formic acid. The total number of unique, non-redundant proteins identified was 395, and the identification was based on 4284 unique peptides. Fourteen CYP450 eluted in fraction 11, eleven in fraction 12, three in fraction 10, three in fraction 9, two in fraction 13, two in fraction 14, and two in fractions 15 and 17. The effective HPLC protein elution fraction was measured as a weighted fraction number average of all fractions using the peptide's LC/MS reconstructed ion chromatogram (RIC) peak heights as the weighing factors as follows: [Fraction number F 1 times the sum of peptide RIC heights of protein "A" in F 1 + ... + Fn multiplied by the sum of peptide RIC heights of protein "A" in Fn ] / [total sum of the LC/MS RIC peak heights for all the peptides of protein "A" in all HPLC protein fractions F1 through Fn]. The following are the most abundant CYP450s, the summed corresponding peptide peak heights in the LC/MS RICs, and the BioworksTM protein scores for experiments conducted with or without (in italics) DMSO and acetonitrile in the protein samples: Cyp2D10, LC/MS integrated RIC peptide peak height sum of 13,285,162 (2,314,728), protein score 166 (138); Cyp2F2, height total 5,190,395 (1,880,942), score 224 (176); Cyp2D9, height total 5,258,772 (704,085), score 110 (78); Cyp2E1, height total 4,203,525 (467,353), score 126 (100), Cyp2D22, height total 2,788,237 (1,414,142), score 110 (56). Average Protein Score for the 37 P450s was 78 in and 69 for the 25 CYP450s identified without the DMSO/ACN presence in the microsomal protein sample.