| Metadata |
| datasetIdentifier | PASS00542 |
| datasetType | SRM |
| submitter | Habtom Ressom <hwr@georgetown.edu> |
| submitter_organization | Georgetown University Medical Center |
| lab_head_full_name | Habtom W. Ressom |
| lab_head_email | hwr@georgetown.edu |
| lab_head_organization | Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center |
| lab_head_country | USA |
| datasetTag | RessomLab_Serum_MRM |
| datasetTitle | Human Serum LC-MRM-MS data targeted 101 proteins on 205 samples collected from two cohorts of HCC and cirrhosis patients. |
| publicReleaseDate | 2014-12-28 00:00:00 |
| finalizedDate | 2014-07-24 13:51:27 |
| summary | In the present study, we used sera from 205 patients representing two cohorts for biomarker discovery using label-free proteomic analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We performed first untargeted proteomic analysis of sera to identify candidate proteins with statistically significant difference between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. These proteins were then evaluated through targeted quantitation by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. |
| contributors | Habtom W. Ressom, Tsung-Heng Tsai, Cristina Di Poto, Minkun Wang, Yehia Mechref, Ehwang Song, Rui Zhu, Yue Luo, Rency S. Varghese, Mahlet G. Tadesse, Dina Hazem Ziada, Chirag S. Desai, Kirti Shetty |
| publication | unpublished |
| growth | |
| treatment | |
| extraction | Sera were subjected to depletion using Agilent Plasma 7 Multiple Affinity Removal Spin Cartridge from Agilent Technologies (Santa Clara, CA). This cartridge depletes the seven most abundant human serum proteins, namely albumin, IgG, antitrypsin, IgA, transferrins, haptoglobin and fibrinogen. A 15-µl aliquot of serum was depleted as stated in the protocol recommended by the manufacturer. The buffer of the depleted sample was exchanged into of 50 mM ammonium bicarbonate (pH 8.0) using 3 kDa MWCO Amicon Ultra 0.5 mL centrifugal filters from Sigma-Aldrich. This buffer is needed for tryptic digestion. |
| separation | Prior to trypsin digestion, the protein concentration of depleted serum was determined through micro BCA protein assay (Thermo Scientific/Pierce, Rockford, IL). The bovine serum albumin (BSA) standard stock solution of 2.0 mg/ml concentration provided in the micro BCA assay kit was used to prepare a set of diluted BSA standard samples with concentrations of 200 µg/ml, 40 µg/ml, 20 µg/ml, 10 µg/ml, 5 µg/ml, 2.5 µg/ml and 1 µg/ml. 50 mM ammonium bicarbonate was used to prepare the BSA standard samples. The micro BCA working reagent required for the assay was prepared by mixing reagents A, B, and C (provided by the vendor) at a ratio of 50:48:3. Next, a 15-µl aliquot of depleted serum was diluted in 135 µl of 50 mM ammonium bicarbonate. BSA standard samples and depleted serum were then mixed with 150-µl aliquots of the working buffer and transferred to a 96-Well Plate prior to incubation at 37ºC for 2 hours. The concentration was then measured at 620 nm wavelength on Multiskan plate-reader (Thermo Scientific, Rockford, IL). |
| digestion | A 20-µg aliquot of depleted serum proteins was transferred to an Eppendorf tube, to which100-µl of 50 mM ammonium bicarbonate was then added. Thermal denaturation was performed at 65 ºC for 10 min. DTT and IAA solutions are prepared in 50 mM ammonium bicarbonate. Sample was reduced by adding a 1.25-µl aliquot of 200 mM DTT solution and incubated at 60 ºC for 45 min. The reduced proteins were then alkylated by adding of a 5-µl aliquot of 200 mM of IAA and incubated at 37.5 ºC for 45 min. A second 1.25-µl aliquot of 200 mM DTT was added and followed by incubation at 37.5 ºC for 30 min to consume excess IAA. A 0.8-µg aliquot of trypsin was added to the sample (enzyme/substrate ratio of 1:25 w/w), and then incubated at 37.5 ºC overnight. This was followed by microwave-assisted digestion at 45 ºC for 30 min at the power of 50 W. The enzymatic digestion was quenched by adding 0.5-µl neat FA to the samples. Then, the samples were speed-vacuum dried and re-suspended in 0.1% FA prior to LC-MS/MS and LC-MRM-MS analyses. |
| acquisition | The LC conditions described previously in the untargeted analysis were used here for targeted quantitation by MRM on the TSQ Vantage mass spectrometer, which was operated in positive mode with an ESI voltage of 1800V. Data independent acquisition mode was used for MRM experiment. Predefined precursor and transition ions were monitored to specifically select targeted peptides corresponding to each candidate protein with 10.0 sec chromatogram filter peak width. The MRM experiments were performed at a cycle time of 5.0 sec and a Q1 peak width of 0.70 min. The collision energy value for each targeted peptide is predicted by Pinpoint (Thermo Scientific, San Jose, CA) with a collision gas pressure of 1.5 mTorr in Q2. |
| informatics | The LC-MRM-MS data were analyzed using Skyline (version 2.5.0.6079). Peptide search results from Andromeda, i.e., msms.txt and mqpar.xml, were used to recognize the monitored transitions from LC-MRM-MS data. The Skyline determined the RT location and integration boundaries for each peptide in each run independently. By comparing the same peptide across runs, we adjusted the RT location and integration boundaries to exclude interfering regions. We selected the peak closest to the RT center of segment if multiple peaks were detected. Each protein’s intensity was quantitated using the summation of intensities from its corresponding six transitions. The difference between total area and background was assigned to quantify a transition. Prior to the statistical analysis, the quantitated protein intensities were log-transformed and normalized by the summed intensity. The most relevant proteins with differential abundance between HCC cases and cirrhotic controls were selected using t-test, and the associated p-values were adjusted based on multiple testing correction (FDR <0.05). |
| instruments | TSQ Vantage |
| species | Human |
| massModifications | static: C+57.021464, variable:M+15.994915, Acetyl+42.010565 |
