4800 plus maldi tof tof mass spectrometer

Differentially expressed protein spots were excised manually from the 2D-DIGE gels, which were previously stained with Oriole^TM fluorescent gel stain (Bio-Rad, Hercules, CA, USA). Additionally, a preparative gel using 400 µg of total protein was prepared for the identification of small or low-abundance proteins, using the same electrophoretic parameters. Spots were automatically digested with the Ettan Digester workstation (GE Healthcare) and identified at the Proteomic Unit of Hospital Nacional de Parapléjicos. The digestion was performed according to Shevchenko et al. (1996) (link) with minor modifications and, after digestion at 37°C overnight, the peptides were extracted with 60% acetonitrile (ACN) in 0.1% formic acid. Samples were dried in a speedvac and resuspended in 98% water with 0.1% formic acid and 2% ACN. An aliquot of each digestion was mixed with an aliquot of the matrix solution (3 mg/ml matrix α-cyano-4-hydroxycinnamic acid in 30% ACN, 15% 2-propanol and 0.1% trifluoroacetic acid), and this was pipetted directly on a 384 Opti-TOF 123×81 mm stainless steel sample plate and analyzed on a 4800 Plus MALDI TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA).

Immunoprecipitation was performed using protein A Dynabeads (Life Technologies) coated with LILR-Fc protein. 50μl PBS-T-washed Dynabeads were loaded with LILR-Fc following incubation with 50μl of 250μg/ml freshly made LILR-Fc at 4°C for 1 hour followed by two washes with PBS-T. The construction and preparation of the LILR-Fc protein is described in the Supplementary Material.
Cell lysates (100μl) were precleared by incubation at 4°C for 1 hour with 25μl protein A Dynabeads. 50μl of LILR-Fc loaded Dynabeads were then added to the precleared lysate for 1hr at 4°C. Beads were washed thrice in cold PBS-T and resuspended with LDS loading buffer (30mM Glycine pH2.8, 1x Nupage LDS buffer and reducing agent [Life Technologies]). Samples were denatured at 70°C for 10 min, before loading onto a Nupage 4-12% Bis-Tris SDS PAGE gel (Life Technologies) alongside SeeBlue II marker (Life Technologies). Gels were run in MOPs buffer (Life Technologies) for 45 minutes at 200V. Protein bands were visualised following Coomassie Blue staining.
Protein bands in gels were identified by fingerprinting of tryptic peptide using an Applied Biosystems 4800 Plus MALDI-TOF-TOF Mass Spectrometer in CIMR/IMS Proteomics Facility (CIPF) of the Cambridge Institute for Medical Research.

In-gel digestion of protein was done according to the established protocol52 (link). The peptides solution with CHCA matrix solution were analyzed on 4800 plus MALDI-TOF/TOF mass spectrometer (Applied Biosystems, USA) in the m/z range 800–3500. The combined PMF search was carried out using GPS Explorer™ software with the MASCOT search engine against C. parasitica database v1.0 (39 genome scaffolds totaling 43.9 MB, 11,184 gene models) from JGI website (http://genomeportal.jgi-psf.org/Crypa1/Crypa1.download.ftp.html) on a local server.