Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.; Carter-Su, Christin

Phosphotyrosine-containing peptides were isolated by immunoaffinity purification with immobilized ␣PY (PhosphoScan P-Tyr-100; Cell Signaling Technology, Danvers, MA) according to the vendor's protocol. Peptides that did not bind to the immobilized ␣PY were further fractionated by SCX HPLC, and the fractions were enriched for Ser-/Thr-phosphopeptides using ZrO 2 (25) . The peptides that bound to and eluted from the immobilized ␣PY, the SCX fractions enriched for Ser-/Thrphosphopeptides using ZrO 2 , and the peptides which did not bind to the ZrO 2 were analyzed by LC-MSMS on a nano LC system (Eksigent Technologies, Dublin, CA) interfaced with a linear trap quadrupole (LTQ)-Orbitrap XL mass spectrometer (Thermo-Fisher Scientific). The Thermo-Fisher Orbitrap was used for this study because its high mass accuracy and resolution reduces the false positives for peptide identification. Peptides were separated using a 75 m ϫ 15 cm column in-house packed with 3 m C18 resin (Sepax HP-C18) over a 120-min gradient of 10 -45% acetonitrile with 0.1% formic acid at a flow rate of 250 nl/min. Analytes were sprayed into the mass spectrometer via a chip-based nanoelectrospray source (Advion Triversa Nanomate) in positive ion mode. The LTQ-Orbitrap was operated in data-dependent mode by alternating single MS scans (300 -1700 m/z) in the Orbitrap analyzer and sequential MSMS scans in the LTQ for the seven most intense ions from each MS survey scan. MS scans were acquired with the resolution (mass/ peak width at half maximum) set at 60,000 at 400 m/z and an automatic gain control target of 1 ϫ 10 6 . MSMS scans were triggered on ions with signal intensity above 500. Recurring precursor ions were dynamically excluded for 30 sec. By applying charge-state monitoring, ions with 1ϩ or unassigned charge states were rejected to increase the fraction of ions producing useful fragmentation. The raw data files from LC-MSMS experiments were analyzed with MaxQuant software (version 1.0.13.13) (38) for peptide identification and quantification. Peak lists were generated from the raw data files using the Quant module in the MaxQuant program and searched by Mascot (39) against a concatenated target-decoy database of IPI-mouse version 3.63. Carbamylation (N terminus), oxidation (Met), and phosphorylation (Ser, Thr, Tyr) were searched as variable modifications. Carbamidomethylation (Cys) was specified as a fixed modification. Search results were maintained by applying 1% false discovery rate for peptide, protein, and site identification. Phosphopeptide pairs that have a normalized SILAC ratio with a significance B less than 0.05 (P value calculated by Max-Quant) were considered to have a statistically significant change in the level of phosphorylation. B less than 0.05 corresponded to a GH-dependent increase of approximately 50% or decrease of about 20% (exact percentage depended on the trial, Supplemental Tables 3-5 published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org).