Here, we provide two kinds of standard data for users to test our tool.
The first data set consists of four samples prepared from the mixture of 10 standard proteins. The four samples were separately digested, labeled with the four isobaric reagents 1(114): 2(115): 1(116): 0.5(117), and then combined and analyzed by LC-MS/MS (Q-STAR, Applied Biosystems, USA ). The second data set consists of two identical protein mixtures prepared from human Jurkat T cells. The cell lysate was first fractionated with SDS-PAGE. One SDS-PAGE band was excised, tryptic digested, and divided into two sets, which were labeled with iTRAQ reagents 114 and 116, respectively. The expected protein expression level in both sets is 1:1. The details of the experiment information related to these two data sets are given below.
We purchased the following chemicals for the experiment: RPMI medium 1640 from HyClone (Logan, UT); fetal bovine serum (FBS) from Gibcobrl (Grand Island, NY); Trypsin from Promega (Madison, WI); Tris (2-carboxyethyl) phoshine (TCEP) from Fisher Scientific Corp. (Pittsburgh, PA); the BCA protein assay kit from Pierce (Rockford, IL); the methyl methanethiosulfonate (MMTS) from Applied Biosystems (Foster City, CA); and Tris, SDS, Triton X-100, trifluoroacetic acid (TFA), formic acid, and acetronitrile from Sigma-Aldrich (St. Louis, MO).
Preparation of Protein Standards
The protein standard was derived from 10 proteins in various concentrations, prepared by diluting the standard protein mixture containing 1 mg/ml of bovine serum albumin, enolase 1, trypsinogen, carbonic anhydrase, myoglobin, concanavalin A, lysozyme C, and beta-casein (all purchased from Sigma, St. Louis, MO); and alcohol dehydrogenase and ovalbumin (both purchased from Calbiochem-Novabiochem Corp., San Diego, CA).
Cell Culture and Lysis
Jurkat T cells were cultured in a RPMI medium supplemented with 10% fetal calf serum. The cells were harvested, washed and aliquoted (about ~ 2 x 107 cells) and suspended in a lysis buffer made up of 50 mM Tris at pH 8.0, 0.1% SDS, and 0.02% Triton X-100. The individual suspensions were lysed by placing them in a sonic water bath for 30 minutes followed by subjection to three freeze (liquid N 2 ) and thaw cycles. Cellular debris was removed by centrifugation at 17,000 g for 45-60 minutes. The total protein concentration of each cell lysate was determined by BCA assay. The protein concentration was adjusted to approximately 1 mg/ml using 50 mM of Tris buffer containing 0.1% SDS.
Fractionation and Tryptic Digestion
Approximately 0.6 mg of the total protein was reduced with TCEP and alkylated with MMTS. SDS-PAGE was then applied to the cell lysates to fractionate proteins using 5-15% gradient gel in a Bio-Rad gel electrophoresis system (Hercules, CA). After staining with coomassie blue followed by destaining with 7% acetic acid in 25 % methanol, the molecular weight region of about 50-90 kDa was excised and further diced into 1 × 1 mm pieces. It was then subjected to in-gel tryptic digestion at 37 o C overnight. The digested peptide mixture was extracted from gel pieces by removing the supernatant with 0.1 % TFA in 50 % (v/v) acetonitrile treated with sonication.
The digested peptides were first desalted with C18 macrotrap (Michrom, Auburn ,CA). The volume of eluted peptide solution was about 200 μl. The resulting peptide solution was divided into two parts: 100 μl was labeled with iTRAQ 114 reagent, and the other 100 μl was labeled with iTRAQ 115 reagent according to the manufacturer's protocol (Applied Biosystems iTRAQ reagents, Applied Biosystems, Foster City, CA).
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
The labeled peptide sample was loaded and sequentially analyzed by μLC-MS/MS. Samples were injected into a 2 cm × 100 μm capillary trap column packed in-house (Magic C 18 , Michrom BioResources, Inc., Auburn, CA). Peptides were separated by a 10 cm × 75 μm capillary column, also packed in-house (Magic C 18 , Michrom BioResources, Inc., Auburn, CA), and eluted with a linear gradient of 5-35% B for 60 min at ~200-300 nl/min (Buffer A: 0.1 % acetic acid in H 2 O; Buffer B: 0.1 % acetic acid in acetonitrile). An HP1100 solvent delivery system (Hewlett Packard, Palo Alto, CA) was used with pre-column flow splitting, and a QSTAR Pulsar i mass spectrometer (Applied Biosystems, Foster City, CA) was used for all analyses. Peptide fragmentation by collision-induced dissociation was performed automatically using the information-dependent option, and the resultant MS/MS spectra were recorded.
Download Supplementary Information of Test Data
Supplementary Table 1. Detailed description of protein and peptide identification and quantification results of 10 standard proteins:
Supplementary Table 2. Detailed description of protein and peptide identification and quantification results in Jurkat cell.
Download Sample Test Data
STD experiement: (Will be available soon.)