Alpha cyano 4 hydroxycinnamic acid matrix

MALDI-TOF MS is a powerful technique that has been used for protein identification from different biological samples [16 ]. The resulting digests were analyzed by 4800 Plus MALDI-TOF/TOF analyzer ABSciex (Applied Biosystems, Foster City, CA), as previously described [11 (link)]. Briefly, the samples were eluted in alpha-cyano-4-hydroxycinnamic acid matrix (Sigma-Aldrich, St Louis, MO). One μl aliquots was spotted directly onto a 384-well Opti-TOF MALDI stainless steel plates (AB Sciex, Framingham, MA) and allowed to air-dry at room temperature. Ions were generated by pulsing the mixture with a nitrogen laser (Laser energy at 4000 nanojoules). The collected spectra were analyzed using ProteinPilot™ software (Applied Biosystems) by searching the mouse and S. mansoni Swiss-Prot protein databases. The enzyme of cleavage was trypsin, fixed modification was carbamidomethyl, and variable modification was oxidation. The confidence threshold for protein identification was set to 95%.

From each TMA, a section of 4 μm was adhered to an indium-tin-oxide (ITO) slide (Bruker Daltonics, Bremen, Germany). Sample preparation has previously been described in detail [15 (link),16 (link)]. Briefly, sample slides were heated to 80 °C prior to dewaxing with xylene (Carl Roth GmbH, Karlsruhe, Germany), and subsequent rehydration with increasingly concentrated ethanol washes (Carl Roth GmbH, Karlsruhe, Germany). Afterward, the samples were subjected to heat-induced antigen retrieval in MilliQ water at 95 °C for 20 min. A trypsin (Promega, Mannheim, Germany) solution was prepared in 40 mM ammonium bicarbonate (Sigma-Aldrich Chemie GmbH, Munich, Germany) to a final concentration of 0.1 µg/µL. The enzyme solution was sprayed with an automatic sprayer (TM Sprayer, HTX Technologies, Chapel Hill, NC, USA) in 16 cycles with a fixed spraying flow of 150 μL/min. On-tissue digestion was carried out for 2 h at a controlled temperature of 50 °C. Following digestion, four cycles of matrix solution (10 mg/mL of alpha-cyano-4-hydroxycinnamic acid matrix (Sigma-Aldrich Chemie GmbH, Munich, Germany) in 70% acetonitrile aqueous solution with 1% trifluoracetic acid (Carl Roth GmbH, Karlsruhe, Germany)) were deposited with a defined flow of 120 μL/min and a temperature of 75 °C.

Isolated gel spots representing proteins of interest were processed as follows for MS-based identification. Trypsin (Promega) digestion was performed in the presence of reduction (dithiothreitol (Bio-Rad)) and alkylation (iodoacetamide (Sigma)) conditions at 37°C as described (103 (link)). This was followed by desalting and concentrating of samples using Ziptips (Millipore). Samples were then applied to a target plate with alpha-cyano-4-hydroxycinnamic acid matrix (Sigma). Data were collected on a 4800 MALDI TOF/TOF Analyzer (ABSciex) in positive ion, reflector mode over a mass range of 850–4000 m/z. Select peaks were further analyzed by tandem mass spectrometry (MSMS) at 1 kV with default calibration. The combined MS and MSMS data were submitted to Mascot v2.4 (Matrix Science) and searched against the NCBI (Mus musculus taxonomy), with Trypsin specificity, 300 ppm mass tolerance, 0.3 Da MSMS tolerance and variable modifications: for carbamidomethylation (C) and oxidation (M). Proteins were identified based on database matches of ≥99% confidence, including three MSMS matches >99% confidence.