Furthermore, the combined capture of multiple proteins from the same sample saves precious patient material and allows faster data evaluation. Our method can be applied in large clinical studies for biomarker development in various disease contexts. technical standards. Negative controls, consisting of PBS, were included at random locations in the sample plate layout (= 20). Immunoaffinity Enrichment of Immunoglobulins from Serum Using a Microlab STAR liquid handling robot (Hamilton, Bonaduz, Switzerland), 5.4 L of serum were pipetted into V-bottom 96-well plates (Greiner Bio-One B.V., Alphen a/d Rijn, The Netherlands) containing 130 L 35 mM phosphate-buffered saline (PBS; pH 7.6, made in-house from 5.7 g Na2HPO4/L of water, 2H2O, 0.5 g KH2PO4/L of water, and 8.5 g NaCl/L of water). Ig enrichment was performed similar to Selman et al. with the following modifications.21 Per sample, 40 L Ureidopropionic acid slurry was used, containing 0.2 L CaptureSelect FcXL Affinity Matrix beads (absolute theoretical capacity for total IgG: 5 g) and 5 L CaptureSelect IgA Affinity Matrix beads (absolute theoretical capacity for IgA: 40 g) in PBS. The bead slurry was applied to 96-well filter plates (10 m pore size, Orochem, Ureidopropionic acid Naperville, IL) and washed three times with 200 L PBS using a vacuum manifold (50 kPa pressure gradient). To prevent drying of the beads 30 L of PBS were added to each well. For combined IgG and IgA capturing, 125 L of diluted sample (corresponding to 5 L serum) were added per well, followed by 1 h Rabbit Polyclonal to OR52E5 incubation at room temperature with agitation. Using a vacuum manifold, the beads were washed three times with 200 L PBS and three times with 200 L water, prior to centrifugation for 1 min at 911 and dried by vacuum centrifugation for 2.5 h at 60 C. Glycopeptide Preparation Dried eluates were dissolved in 10 L reduction-alkylation buffer (100 mM tris(hydroxymethyl)aminomethane, 1% (w/v) SDC, 10 mM TCEP and 40 mM CAA) and sealed with VersiCap Mat flat cap strips (Thermo Fisher Scientific) followed by 5 min of shaking at 500 rpm with 1.5 mm orbit. For one-step denaturation, reduction, and alkylation the samples were incubated for 5 min at 95 C and cooled to room temperature in a 2720 Thermal Cycler (Thermo Fisher Scientific). For digestion, 50 L of digestion buffer (0.004 g/L sequencing grade modified trypsin in 50 mM ammonium bicarbonate (pH 8.5) was added to each sample. The plate was closed with VersiCap Mat flat cap strips, followed by 5 min shaking at 1000 rpm and overnight incubation at 37 C. On the next day, 1.2 L of concentrated formic acid was added to each sample, followed by centrifugation for 45 min at 2800 for the acid precipitation of SDC. Using a semiautomated pipetting system Liquidator 96 (Mettler Toledo, s-Hertogenbosch, Netherlands), 40 L of supernatant containing Ig (glyco-)peptides were transferred to a V-bottom 96-well plate (Greiner). The plate was sealed and stored at ?20 C prior to MS analysis. Protein and Glycopeptide Ureidopropionic acid Identification by MS/MS (Glyco-)peptides were identified in one serum sample pooled from a subset of the clinical cohort described above. To this end, an Easy nLC 1200 gradient system (Thermo Fisher Scientific), and an Orbitrap Fusion Lumos Tribrid MS were used (Thermo Fisher Scientific; Supporting Information (SI) Tables S1 and S2 and Figure S1). The tryptic digest sample was injected into a homemade precolumn (15 mm 100 m; Reprosil-Pur C18-AQ 3 m, Dr. Maisch, Ammerbuch, Germany). Separation was performed via an analytical nanoLC column (15 cm 75 m; Reprosil-Pur C18-AQ 3 m; drawn to a spray tip of 5 m at the end of the column) with a solvent B (20/80/0.1, water/acetonitrile/FA, v/v/v) gradient from 10 to 40% in 20 min (solvent A: 0.1% formic acid in water). After electrospray ionization, a data-dependent tandem MS analysis was performed where a full MS1 scan within 400C1500 was collected with a resolution of 120?000, at an automatic gain control (AGC) target of 400?000 and with a maximum fill time of Ureidopropionic acid 50 ms. The most abundant ions per 3 s were fragmented with higher-energy C trap dissociation (HCD, 32 normalized collision energy). Dynamic exclusion.