Project PXD000004

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Summary

Title

Identification and quantification of human postmortem frontal cortex proteome with a SILAM mouse brain standard

Description

Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).

Sample Processing Protocol

See details in reference PMID : 22942359

Data Processing Protocol

See details in reference PMID : 22942359

Contact

Matthew MacDonald, Psychiatry

Submission Date

25/06/2012

Publication Date

10/12/2012

Tissue

brain

Experiment Type

Bottom-up proteomics

Assay count

5

Publication

    MacDonald ML, Ciccimaro E, Prakash A, Banerjee A, Seeholzer SH, Blair IA, Hahn CG; Biochemical fractionation and stable isotope dilution liquid chromatography-mass spectrometry for targeted and microdomain-specific protein quantification in human postmortem brain tissue., Mol Cell Proteomics, 2012 Dec, 11, 12, 1670-81, PubMed: 22942359

Assay

Showing 1 - 5 of 5 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 26881 Workflow for quantifying SILAC (Arg10, Lys 6) duplex labeled samples using Sequest. All modifications are set in the quantification method as well as in the Sequest search node. If you use other SILAC labels please change that in the quantification method as well as in the Sequest search node. 9119 514966 100949 92434 77144
2 26882 Workflow for quantifying SILAC (Arg10, Lys 6) duplex labeled samples using Sequest. All modifications are set in the quantification method as well as in the Sequest search node. If you use other SILAC labels please change that in the quantification method as well as in the Sequest search node. 8537 619256 120087 98093 85951
3 26883 Workflow for quantifying SILAC (Arg10, Lys 6) duplex labeled samples using Sequest. All modifications are set in the quantification method as well as in the Sequest search node. If you use other SILAC labels please change that in the quantification method as well as in the Sequest search node. 3939 312393 52183 78247 63278
4 26884 Workflow for quantifying SILAC (Arg10, Lys 6) duplex labeled samples using Sequest. All modifications are set in the quantification method as well as in the Sequest search node. If you use other SILAC labels please change that in the quantification method as well as in the Sequest search node. 2622 190532 29566 65204 47397
5 26885 Workflow for quantifying SILAC (Arg10, Lys 6) duplex labeled samples using Sequest. All modifications are set in the quantification method as well as in the Sequest search node. If you use other SILAC labels please change that in the quantification method as well as in the Sequest search node. 1950 133394 19320 56508 36785