Qstar xl

Coomassie-stained proteins within the gel pieces were reduced, carboxyamidomethylated, and digested to peptides using trypsin on a MassPrepStation (Waters, Manchester, UK). The resulting peptides were applied to a LC-MS/MS column. For LC-MS/MS, the reverse phase liquid chromatographic separation of peptides was achieved with a PepMap C18 reverse phase, 75 mm i.d., 15-cm column (LC Packings, Amsterdam) on a capillary LC system (Waters) attached to QTof2 (Waters) mass spectrometer or the same column attached to a Dionex Dual Gradient LC system attached to a QSTAR XL (Applied Biosystems, Framingham, MA, USA). The MS/MS fragmentation data achieved was used to search the National Center for Biotechnology Information database using the MASCOT search engine (http://www.matrixscience.com).

Total proteins from SILAC-labeling HEK293 cells and RCC, PKT tissues were prepared according to our previous reports [21 (link), 22 (link)]. 30 µg cellular proteins from HEK293 cells were respectively mixed with equal proteins from RCTs and PKTs, and two group of protein mixture was isolated by SDS-PGAE. The 110-kDa band was cut to digest and peptides were identified by LC-nanospray-tandem mass spectrometry (MS/MS) using a QSTAR XL mass spectrometer (Applied Biosystems, USA). The relative protein expression level was quantified by tracking pairs of labeling and unlabeling peptides from the MS spectra.

Rhode Island Hospital COBRE Proteomics Core facility performed all mass spectrometry analysis by nano-LC-ESI-MS/MS using an Ultimate3000 nano-LC system (Dionex) controlled with Chromeleon software coupled to a QSTAR XL (Applied Biosystems, Concord, Ontario, CA) mass spectrometer. Tryptic digests were fractionated by reverse-phase chromatography using a C-18 PepMap 100 column (75 um id x 15 mm, 3um particle size, LC Packings/Dionex, Sunnyvale, CA) operating at a flow of 300 nL/min. Over a 40 min time period, a linear separation gradient was applied starting at 5 % (v/v) CAN in 0.1 % (v/v) formic acid (Buffer A) to 95 % (v/v) ACN in 0.1 % (v/v) formic acid (Buffer B). ESI was used to introduce the column elutate directly into the mass spectrometer.
Candidate ions were selected and fragmented using a standard information dependent acquisition (IDA) method. During MS/MS scans, one-second MS scans (range between 350 and 1800 Thompson, Thompson (Th) = Da/z) were used to identify candidates for fragmentation. MS/MS scans (2 s; range between 150 and 1800 Th) were collected up to three times after each survey scan. Candidates considered for fragmentation required an assigned charge in the range of +2 or +4.

The samples marked with iTRAQ were dissolved in cation-exchange (SCX) buffer for SCX separation. The four samples containing mesenchymal proteins from NGM, AH, GPDAC and LMGAC tissues were mixed and loaded into a polysulfoethyl column and segregated by an LC-20AD high-performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan) following the manufacturer's instructions. Next, the products were concentrated by vacuum centrifugation for reverse-phase HPLC-mass spectrometry (MS) analysis. The samples were dissolved in 50 µl of 5% ACN containing 0.1% FA and loaded into a Zorbax 300SB-C18 column (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. The data were analyzed using QSTAR-XL (Applied Biosystems, Thermo Fisher Scientific) and tandem MS (MS/MS). Finally, the IPI human database (version 3.45, URL: http://www.ebi.ac.uk/IPI) was searched for protein information, the confidence level was set as >95%, and the ion peak areas of m/z 114 and 118 were integrated.

A detailed protocol for proteomics analysis was followed as previously described [15 (link)]. Briefly, iTRAQ labeling (Applied Biosystems) coupled with online two-dimensional Nano LC/MS/MS system (2D-nanoLC-MS/MS) (Agilent, Waldbronn, Germany) was used; protein (100 μg) from each sample was denatured, alkylated, and digested before being labeled with iTRAQ reagents and mixed. After being cleaned, desalted, and vacuum-dried, the mixed sample was analyzed using the On-line two-dimensional Nano LC/MS/MS on a Nano-HPLC system coupled to a hybrid Q-TOF mass spectrometer (QSTAR XL, Applied Biosystems) equipped with a Nano-ESI source (Applied Biosystems) and a Nano-ESI needle (Picotip, FS360-50- 20; New Objective Inc., Woburn, MA). The ProteinPilot™ Software 3.0 (revision 114732) was used to analyze the differential expressed protein. A ratio more than 1.5-fold or less than 0.66-fold was deemed significant. Using the Software DAVID (http://david.abcc.ncifcrf.gov/) coupled with wego (http://wego.genomics.org.cn/cgi-bin/wego/index.pl), the GO term analysis was completed and the differential expressed protein of each group was determined. STRING (V9.1) software was then used to evaluate the network with crosstalk key molecules and with transcription factors.

The rSK was identified by MALDI-TOF mass spectrometry as previously described [18 (link)]. The predicted protein band on SDS-PAGE was cut out and the target protein was digested by trypsin treatment into small peptide fragments. The mixture of peptides was analyzed on nano-LC liquid chromatography and ionized by the ESI (electrospray ionization). The mass spectra were obtained by QSTAR XL mass spectrometer (Applied Biosystems, MDS SCIEX, Canada) with a nano-ESI ion source. Protein fragments were identified by the Mascot v1.8 Search Software from the database (NCBInr, SwissProt). Peptide fragments showing ion scores above 42 were identified uniquely or high-similarly with P < 0.05.