Objective

The University of Washington, Department of Laboratory Medicine recently validated a sensitive LC-MS assay to quantify C-peptide in human serum.  Multi-plexing additional diabetes biomarkers in this established assay is desirable from scientific, clinical, and efficiency perspectives.  With this motivation, a data-independent-acquisition (DIA) mass spectrometry (MS) experiment was performed to identify proteins of interest present in the prepared C-peptide assay sample.

Sample preparation

Publication is pending for the full detailed LC-MS C-peptide assay development, final method, and validation results.  Briefly, high abundance proteins are precipitated from 200 µL of serum by adding 400 µL of acetonitrile, and supernatants are collected using a Pall AcroPrep 0.45 µm PTFE filter plate by centrifugation for 10 min at 4000 rpm.  The supernatants are dried down in a speedvac at room temperature overnight.  Samples are resuspended in 90 mM ammonium bicarbonate, 10% acetonitrile, then acidified to 3% formic acid final, and subjected to solid-phase extraction using Waters Oasis MAX µElution plates per manufacturer’s recommended conditions.  Eluates are evaporated to dryness under nitrogen in a TurboVac, then resuspended in 90 mM ammonium bicarbonate, 10 mM DTT, 10% acetonitrile and reduced in a Thermomixer for 1 h at 60°C and 900 rpm.  Samples are alkylated with 28 mM iodoacetamide in the dark for 30 min, then digested with 140 µg of endoproteinase Glu-C for 2 h at 30°C and 900 rpm, and finally acidified with formic acid.

For the purposes of this study, human serum pool from Golden West (SP2001) was prepared in 12 replicate wells on two different days.  Prior to digestion, protein concentration was determined by Bradford assay for one representative sample on each day.  The digested replicate samples were pooled (N=22), aliquoted and stored at -80°C prior to MS analysis.  Bradford assay results were used to target a 1 µg injection amount.

LC-MS Analysis

Peptides were analyzed with a Thermo Scientific EASY-nanoLC 1000 system coupled to a Thermo Q-Exactive Plus tandem mass spectrometer.  New Objective PicoTip PicoFrit Emitters (75 µm inner diameter) columns were packed with 3 μm ReproSil-Pur 120 C18-AQ resin (Dr. Maisch) to approximately 300 mm, coupled with a Kasil fritted trap column created from 150 μm inner diameter fused silica packed with Phenomenex Jupiter 4µm Proteo 90Å resin to approximately 25 mm.  Mobile phase A was 2% acetonitrile, 0.1% formic acid in water, while mobile phase B was 0.1% formic acid in 98% acetonitrile.  Peptides were resolved using a 90 min gradient from 5 to 45% B, followed by a 34 min wash at a flow rate of 250 nL/min.  To create a DIA chromatogram library, eight injections with 4 m/z DIA spectra were collected (4 m/z precursor isolation windows at 15,000 resolution, AGC target 1e6, maximum inject time 55 ms, 27 NCE) using a staggered (also referred to as overlapping) window pattern from narrow mass ranges using window placements optimized by Skyline (i.e., 398.43–502.48, 498.48–602.52, 598.52–702.57, 698.57–802.61, 798.61–902.66, 898.6–1002.70 m/z, 998.61–1102.75, and 1098.75–1202.80 m/z).  Precursor spectra, a narrow spectrum matching the range (i.e., 390–510, 490–610, 590–710, 690–810, 790–910, 890–1010, 990–1110, and 1090–1210 m/z) using an AGC target of 3e6 and a maximum inject time of 100 ms were interspersed every 26 MS/MS spectra.  For single-injection runs, the Thermo Q-Exactive Plus was configured to acquire 25 x 24 m/z (400-1000 m/z), precursor isolation window DIA spectra (30,000 resolution, AGC target 1e6, maximum inject time 55 ms, 27 NCE) using a staggered window pattern using window placements optimized by Skyline.  Precursor spectra (target range ± 15 m/z at 30,000 resolution, AGC target 3e6, maximum inject time 100 ms) were interspersed every 25 MS/MS spectra.

Data analysis

Staggered DIA data were de-multiplexed with 10 ppm accuracy in ProteoWizard (3.0.19115)Comet was used to search the de-multiplexed 4 m/z DIA mzMLs with partial Glu-C digestion, maximum 4 missed cleavage, dynamic methionine oxidation and static carboxyamidomethylcysteine to generate spectral libraries.  EncyclopeDIA was used to search the resulting de-multiplexed mzMLs, with the default settings: 10 ppm precursor, fragment, and library tolerances, using both B and Y ions, and assuming partial digestion.  EncyclopeDIA search results were filtered to a 1% peptide-level using Percolator 3.1 and then filtered again to a 1% protein-level false discovery rate (FDR) assuming protein grouping parsimony.