| Metadata |
| datasetIdentifier | PASS00616 |
| datasetType | SRM |
| submitter | Christopher Colangelo <christopher.colangelo@yale.edu> |
| submitter_organization | YALE UNIVERSITY |
| lab_head_full_name | Christopher Colangelo |
| lab_head_email | christopher.colangelo@yale.edu |
| lab_head_organization | YALE UNIVERSITY |
| lab_head_country | United States |
| datasetTag | TUPA0001 |
| datasetTitle | Targeted Urine Proteome Assay For Kidney Diseases |
| publicReleaseDate | 2015-11-03 00:00:00 |
| finalizedDate | 2014-11-03 11:38:51 |
| summary | The Targeted Urine Proteome Assay (TUPA) consist of 200 potential urine protein biomarkers. TUPA uses scheduled multiple reaction monitoring (MRM) to determine protein expression based on two “light” and two “heavy”, stable isotope labeled tryptic peptides/protein, monitoring >1,600 transitions and using up to 8 data points (2 transitions/peptide) to determine the actual level of expression of each protein. |
| contributors | Christopher M. Colangelo
KAthryn L. Stone
Lisa Chung
Justion Belcher
Thomas Abbott
Kenneth R. Williams
Jennifer Cantley
LLoyd Cantley
Chirag R. Parikh |
| publication | Christopher M. Colangelo, Kathryn L. Stone, Lisa Chung, Justin Belcher,Thomas Abbott, Kenneth R. Williams, Jennifer Cantley, Lloyd Cantley, Chirag R. Parikh (2014) submitted |
| growth | |
| treatment | |
| extraction | |
| separation | LC-MRM separation was performed on a 5550 QTRAP (AB Sciex, Framingham, MA) instrument with a nanospray source connected to a nanoACQUITY UPLC (Waters, Milford, MA). The mobile phases for LC separation consisted of water (A) and acetonitrile (B) containing 0.1% FA respectively. 3 µg (5 µl of 0.6 µg/µl) of peptides from each sample digest was injected onto a Waters Symmetry C18, 5 µm particle size, 180 µm ID x 20 mm nanoACQUITY UPLC trap column that was connected to a Waters BEH 30 C18, 1.7 µm particle size, 75 µm ID x 150 mm capillary column operated at 45°C. Peptides were trapped for 3 min at 1% B with a flow rate of 5 µl/min. Gradient elution was carried out using a flow rate of 0.5 µl/min with a two-step gradient from 5-40% B over 160 min and then 40-85% B in 3.3 min respectively. |
| digestion | Each of the resulting urine pools with 48 µg (IGF) or 49 µg (DGF) protein were dried in a Speedvac and then dissolved in 8 M urea, 0.3 M triethylammonium bicarbonate (TEAB). The samples were then reduced by adding 2 µl 45 mM dithiothreitol (DTT) and incubating at 37°C for 20 minutes prior to alkylation by the addition of 4 µl of 100 mM iodoacetamide (IAN) and incubation at room temperature for 20 minutes. The samples then were digested first with 2.5 µl of 1 mg/ml Lysyl endopeptidase (catalog # 125-02543 Wako Pure Chemical Industries, Ltd.) for 5 hours at 37°C and then with 2.5 µl of 1 mg/ml trypsin (catalog # V511X Promega) for 16 hours at 37°C for. |
| acquisition | An internal standard containing 399 “heavy” idiotypic peptides (2 peptides/protein for 199 proteins and one peptide corresponding to the BIN1 protein) was added to the tryptic digest of each urine sample. With the MRM assay also monitoring five transitions from each of six “light” peptides from albumin, useful for estimating the relative amount of this typically most abundant urinary protein, the final combined “light” and “heavy” TUPA extended MRM (xMRM) assay contained 1,630 transitions. The MRM method used peak windows of 5 min and a cycle time of 2.5 sec. In the case of hydrophilic peptides that elute before about 20 minutes, the peak window was extended to 400 seconds to allow for the higher variability in the retention times of these peptides. The xMRM assay continuously monitors the primary (1) transition while the secondary transition (2) is only monitored when the primary transition reaches above the threshold value that was set at a value of 200 to be above the noise level. Additionally, the xMRM assay was run with the “timeslip” feature that will extend a scheduled MRM window by an additional half window (2.5 mins) if the peak area threshold is greater than 200 at the end of the first window. All transitions were weighted equally as indicated by the value of “1” in the Weighting column. Other parameters indicated in this table include the declustering potential (DP), entrance potential (EP), collision energy (CE), and Collision Exit Potential (CXP). |
| informatics | The resulting LC-xMRM data was processed and quantitated using MultiQuant™ 2.1 software (research version). The SF2 algorithm (research version) was used to integrate and score peak groups. Data from MultiQuant were exported and uploaded into Yale Protein Expression. |
| instruments | AB Sciex TripleTOF 5600
AB Sciex 5500 QTRAP |
| species | Human |
| massModifications | static: C+57.021464
variable: K+8.014199, R+10.008269 |
