DOI: 10.25345/c5w95120w
title: MassIVE MSV000099698 - KinoPlex: Ultra-Deep Phosphoproteomics in K562 with LC/LC-MS/MS DIA Astral
title: To evaluate the generalizability of KinoPlex to phosphosites absent from all existing databases, we conducted an ultra-deep phosphoproteomic profiling experiment using the K562 cell line. Cells were cultured in standard RPMI + 10% FBS, followed 12-hour serum and nutrient deprivation in HBSS. Cells were then transferred to RPMI + 10% FBS for 30 minutes and subsequently harvested. This transient perturbation was designed to activate nutrient-sensing kinase pathways while also capturing phosphorylation events driven by constitutive oncogenic fusion kinases, notably BCR-ABL, endogenously expressed in K562 cells. Approximately 20 mg of total protein was harvested post-stimulation by lysing cells in 8 M urea supplemented with phosphatase inhibitors. Proteins were purified by methanol-chloroform precipitation, alkylated using iodoacetamide (10mM), reduced with dithiothreitol (15mM), and digested with sequencing-grade trypsin overnight at 1:100 and LysC 1:50. Peptide concentrations were quantified using peptide BCA assay prior to enrichment. Phosphopeptide enrichment was achieved using 20 immobilized metal affinity chromatography (IMAC) columns run in parallel using ThermoFisher High Select Phosphopeptide Enrichment Kit (Cat# A32992), yielding ~400ug of enriched phosphopeptides. Proprietary protocol was adhered to. Following phosphopeptide enrichment, ~400ug of IMAC-purified phosphopeptides were fractionated offline using high-pH reversed-phase high-performance liquid chromatography. Separation was performed using an Agilent 1200 series HPLC system equipped with a C18. Peptides were eluted over a 75-minute gradient using increasing concentrations of acetonitrile (ACN) in 10 mM ammonium bicarbonate, pH 10, across 24 concatenated fractions collected at 2.5-minute intervals. Fractions were subsequently vacuum-dried and reconstituted in 0.1% formic acid for LC-MS/MS analysis. Mass spectrometry was performed using a Thermo Scientific Orbitrap Astral mass spectrometer coupled to an Easy-nLC 1200 nano-flow high-pressure liquid chromatography system. Approximately 500ng of each reconstituted fraction was loaded onto a pre-equilibrated analytical column. Separation was performed on a 50 cm uPAC HPLC column (Thermo Fisher Scientific) at 35C and an Vanquish Neo LC system (Thermo Fisher Scientific). Flow rate was set to 350 nL/min. Mobile phase A consisted of 0.125% formic acid in ACN/water (5:95, v/v). Mobile phase B consisted of 0.125% formic acid in ACN/water (95:5, v/v). When peptides were loaded on the column, mobile phase B increased from 1% to 25% over 22 minutes, increased to 99% over 2 minutes, and stayed at 99% for 6 minutes. Eluting peptides were analyzed by an Orbitrap Astral mass spectrometer operated in data independent acquisition (DIA) mode (Thermo Fisher Scientific). MS1 scan range was set to 380-980 m/z. MS1 normalized AGC target was set to 250%. Isolation window was set to 2 m/z. MS2 scan range was set to 110-2000. MS2 normalized AGC target was set to 200%. HCD normalized collision energy was set to 27%. MS2 normalized AGC target was set to 200%. MS2 maximum injection time was set to 3 ms. Raw data were converted to mzML format and searched using FragPipe (v17.1) with MSFragger (v3.4) against the UniProt human reference proteome. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine and phosphorylation of serine, threonine, and tyrosine were specified as variable modifications. Trypsin/P was set as the protease with up to two missed cleavages allowed. Precursor and fragment mass tolerances were set to +/-20 ppm. Peptide spectral matches (PSMs) were filtered using Philosopher and PeptideProphet with FDR thresholds of 1% at both the PSM and peptide level. Phosphosite localization was refined using PTMProphet, and only confidently localized sites (probability &gt;0.5) were retained for downstream analysis. Sites detected across technical replicates were merged to generate a final non-redundant phosphosite compendium for K562 cells.