Magic c18 packing material

Aleuria aurantia lectin (AAL)-, Sambucus nigra lectin (SNA)-, and wheat germ agglutinin (WGA)-conjugated agarose beads were purchased from Vector Laboratories (Burlingame, CA). Tris (2-carboxythyl) phosphine (TCEP) was purchased from Pierce (Rockford, IL). Sequencing-grade trypsin was from Promega (Madison, WI). Peptide-N-glycosidase F (PNGase) was from ProZyme (San Leandro, CA). Sodium periodate, hydrazide resin, and P6 desalting spin columns were from Bio-Rad (Hercules, CA). C18 and MCX desalting columns were from Waters (Milford, MS). The high-performance liquid chromatography–mass spectrometry (HPLC–MS) platform used in this study includes an Eksigent 2D nanoLC system (Dublin, CA) and a Thermo Scientific Orbitrap Velos mass spectrometer (Waltham, MA). Magic C-18 packing material was from Michrom Bioresources (Auburn, CA), and all other chemicals were purchased from Sigma (St. Louis, MO).

Soluble GST-tagged recombinant PTP-PEST proteins from bacterial lysates were fractionated using glutathione-Agarose prior to mixing with detergent-soluble cell lysates as described previously (23 (link)). Glutathione-bound fractions were resolved on an SDS polyacrylamide gel under reducing conditions. Control and experimental bands were cut from the gel, and subjected to in-gel digestion with trypsin. Peptides were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Peptides were resuspended in 2.5% formic acid (FA), 2.5% acetonitrile (MeCN) and were loaded using a Micro AS autosampler (Thermo Electron) onto a microcapillary column of 100 μm inner diameter packed with 12 cm of reversed-phase Magic C18 packing material (5 μm, 200 Å; Michrom Bioresources, Inc., Auburn, CA, USA). SEQUEST matches were filtered by XCorr scores to a less than 1% false discovery rate when the control proteins were eliminated and protein matches were required to have three spectral matches, no identifiable false positive peptides remained.

Detergent-soluble lysates (1 mg/ml) from primary human GBM cells were used for immunoprecipitation experiments with control rabbit IgG or a rabbit polyclonal antibody directed against the β8 integrin cytoplasmic domain that has been described elsewhere (19 (link)). Antibody complexes were immobilized using Protein G-Sepharose. Immunoprecipitated proteins were resolved on an SDS polyacrylamide gel under reducing conditions. Control and experimental bands were cut from the gel, and subjected to in-gel digestion with Trypsin. Peptides were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as follows. Peptides were resuspended in 2.5% formic acid (FA), 2.5% acetonitrile (MeCN) and were loaded using a Micro AS autosampler (Thermo Electron) onto a microcapillary column of 100 μm inner diameter packed with 12 cm of reversed-phase Magic C18 packing material (5 μm, 200 Å; Michrom Bioresources, Inc., Auburn, CA, USA). SEQUEST matches were filtered by XCorr scores to a less than 1% false discovery rate when the control proteins were eliminated and protein matches were required to have three spectral matches, no identifiable false positive peptides remained.