Nano lc ultra 2d plus

Proteins in the polyacrylamide gels underwent silver staining using a Pierce Silver Stain for Mass Spectrometry Kit (Thermo Scientific). The stained bands were excised and in-gel-digested with trypsin using an In-Gel Tryptic Digestion Kit (Thermo Scientific). The tryptic digests were separated using nanoflow liquid chromatography (Nano-LC-Ultra 2D-plus equipped with cHiPLC Nanoflex [Eksigent, Dublin, CA, USA]). The eluate was directly introduced into a mass spectrometer (TripleTOF 5600+ System coupled to a NanoSpray III source and heated interface [AB SCIEX, Framingham, MA]) and ionized in an electrospray ionization-positive mode. Data were acquired using an information-dependent acquisition method. The acquired datasets were analyzed by ProteinPilot software, version 4.5 beta (AB SCIEX), with the NCBI nr database (June 2016).

Proteins for LC-MS analysis were extracted from FFPE tissue samples (five IgG4-related pancreatitis and three normal pancreas tissue) using the Liquid Tissue MS Protein Prep kit (Expression Pathology Inc, Rockville, MD, USA) [15 (link)]. All cases analyzed for LC-MS were the partial resection for pancreas. For normal pancreas tissues, negative surgical margins taken at resection for pancreatic cancer were selected. Briefly, after deparaffinization, three 0.4 μm thick tissue sections (10 × 10 mm) were dissected using a needle and solubilized in 20 uL of Liquid Tissue buffer and protein digestion was performed with trypsin (Promega Corp, Madison, WI, USA) for 18 h at 37°C. Samples were dried and solubilized in 0.1% formic acid (Wako, Osaka, Japan) and 1 μg aliquots for each sample were separated by nanoflow reversed-phase LC (NanoLC-Ultra 2D-Plus, Eksigent, Dublin, CA, USA) equipped with cHiPLC Nanoflex (Eksigent). Eluted peptides were analyzed by a quadrupole-time-of-flight hybrid mass spectrometer (Triple TOF5600+ system, AB SCIEX, Framingham, MA, USA).

SCX separations were performed on a passivated Waters 600E HPLC system, using a 4.6 × 250 mm PolySULFOETHYL Aspartamide column (PolyLC, Columbia, MD) at a flow rate of 1 ml/min. The gradient was Buffer-A (10 mM ammonium formate, pH 2.7, in 20% acetonitrile/80% water) at 100% (0–22 min following sample injection), 0% → 40% Buffer B (666 mM ammonium formate, pH 2.7, in 20% acetonitrile/80% water, 16–48 min), 40% → 100% Buffer B (48–49 min), then isocratic 100% Buffer B (49–56 min), then at 56 min back to 100% A to re-equilibrate for the next injection. First 26 ml of fractions were combined into one fraction and 32 additional 1 ml fractions were collected and dried down and resuspended in 9 μl of 2% (v/v) acetonitrile, 0.1% (v/v) formic acid, for the 2^nd dimension separation by reverse phase nanoflow LC. The SCX samples were filtered and auto injected using Eksigent NanoLC-Ultra-2D Plus and Eksigent cHiPLC Nanoflex onto a Trap Column (200 μm × 0.5 mm Chrom XP C18-CL 3 μm 120 Å) and eluted through a Nano cHiPLC Column (75 μm × 15 cm Chrom XP C18-CL 3 μm 120 Å). The elution gradient was 95% C (degassed 0.1% formic acid in water)/5% D (degassed 0.1% formic acid in acetonitrile) (300 nl per minute flow rate) to 65% C/35% D in 120 minutes, 15% C/85% D from 120 to 130 minutes, then back to 95% C/5% D from 130–150 min.

The eluted proteins were precipitated with cold acetone, dried and resuspended in 8 M urea/30 mM ammonium bicarbonate. After reduction and alkylation with dithiothreitol and iodoacetamide, the proteins were digested with trypsin overnight, then purified using Pierce C18 spin columns (Thermo Fisher Scientific), dried and resuspended in 0.1% formic acid. The separation was carried out using Nano-LC-Ultra 2D-plus equipped with cHiPLC Nanoflex (Eksigent, Dublin, CA, USA) in trap-and-elute mode, with trap column (200 μm × 0.5 mm ChromXP C18-CL 3 μm 120 Å (Eksigent)) and analytical column (75 μm × 15 cm ChromXP C18-CL 3 μm 120 Å (Eksigent)). The binary gradients used for the separation were as follows: A98%/B2% to A66.8%/B33.2% in 125 min, A66.8%/B33.2% to A2%/B98% in 2 min, A2%/B98% for 5 min, A2%/B98% to A98%/B2% in 0.1 min, and A98%/B2% for 17.9 min, in which 0.1% formic acid/water and 0.1% formic acid/acetonitrile were used as solvents A and B respectively. The flow rate was 300 nL/min. The analytical column temperature was set to 40 °C. The eluates were infused on-line to a mass spectrometer (TripleTOF 5600 + System with NanoSpray III source and heated interface (SCIEX, Framingham, MA, USA)) and ionized in an electrospray ionization-positive mode. Data acquisition was carried out with an information-dependent acquisition method.