4700 proteomics analyzer

Purified enzyme was sent to Sangong Biotech Ltd. (Shanghai, China) for MS and MS/MS analyses with matrix assisted-laser desorption ionization- (MALDI-) time of flight (TOF) and MALDI-TOF-TOF methods.
Protein was digested with trypsin and then analyzed with a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA). The MALDI MS parameters were designed with MS acquisition in the reflector mode, positive ion mode, mass range 850–4,000 (mass/charge (m/z)), and minimum signal/noise (S/N) set at 10 for MS acquisition. Twenty-five of the strongest precursors were chosen for MS/MS, and a minimum S/N 30 was used for MS/MS precursors. The Applied Biosystems GPS Explorer™ v3.6 program was used for a combined search of MS and MS/MS data, with Mascot as the search engine (available at http://www.matrixscience.com/). Searches allowed for carbamidomethylation and monoisotopic oxidation, 100 ppm of peptide mass or parent tolerance, and a maximum of one missed trypsin cleavage. Peptide tolerance and MS/MS tolerance were both 0.5 Da. Proteins with a statistically significant (P < 0.05) protein score were considered as identified with confidence (based on combined mass and mass/mass spectra). Redundancy of proteins that appeared in the database under different names and accession numbers was eliminated.

Total protein extracts from C57Bl/6 mice brain samples were processed as in [7 (link)]. Mass spectrometry was performed on an Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF ion optics as previously described [7 (link)]. All peptide mass values were considered monoisotopic, and a MS mass tolerance was set at 100 ppm. Trypsin was assigned as digestion enzyme of aSyn. A triple miss cleavage was allowed and oxidation of methionyl residues, acetylation of the N-terminus, and acetylation of lysine residues were assumed as variable modifications. All peaks with S/N greater than 5 were included for matching against in silico digestion of corresponding aSyn sequence (Mus muscullus) in mMass software [35 (link)].

Here, we synthesized a cell type‐specific binding of peptides (GGP peptide) for neutrophils and monocytes as conjugator for liposomes (Karathanasis et al, 2009). Briefly, 10.9 mg (5.6 μmol) of GGP peptide was dissolved in 500 μl of dimethyl sulfoxide (DMSO). DSPE‐PEG (2000)‐COOH (10 mg, 3.5 μmol), dicyclohexylcarbodiimide (DCC; 10.8 mg, 53.6 μmol), and 250 μl of pyridine were added to the reaction and incubated for 6 h at ambient temperature. Pyridine was removed by rotary evaporation. Conjugate micelles were formed by hydrating the DMSO solution with 5 ml of deionized water until DMSO reached 10% of the final volume. Unconjugated compounds were removed by dialysis (100 kDa MWCO) against 1 l, 50 mM NaCl (2×), and 1 l of deionized water (2×) followed by lyophilization. Final peptide content was determined using the DC protein assay (Bio‐Rad, Hercules, CA, USA). The final conjugate was characterized by thin‐layer chromatography on pre‐coated plates (silica gel 60 F254) and MALDI‐TOF mass spectroscopy (Applied Biosystems 4700 proteomics analyzer) to confirm molecular mass, with spectrum obtained in negative ion mode in a‐cyano‐4‐hydroxycinnamic acid matrix.

MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) MS analysis was performed on 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, USA). Samples were prepared by acidification with adding 0.1% TFA and subsequent processing via C18 ZipTip column. CHCA (α-cyano-4-hydroxycinnamic acid) matrix was prepared by dissolving 5 mg in 1 ml of 50:50 acetonitrile/water containing 0.1% TFA. Mass spectra for 1–5 kDa were obtained in positive reflectron mode and 5–10 kDa in linear mode.

Protein bands were excised from SDS-PAGE, digested by trypsin and the sample was subjected to MALDI-TOF MS using an Applied Biosystems 4700 proteomics analyzer (Applied Biosystems, CA, USA). Data analysis was performed using the GPS Explorer software and MASCOT with the NCBI database.

The mass-to-charge ratio of MAAs was determined by the Matrix Assisted Laser Desorption/Ionization Time Of Flight Tandem Mass Spectrometry (MALDI-TOF/MS) method using a 4700 Proteomics Analyzer with Denovo Explorer software (Applied Biosystems, Carlsbad, CA, USA). The fractionated MAAs were lyophilized and dissolved in ultra pure water containing 0.1% TFA. Then, the samples were mixed with 5 mg/mL α-cyano-4-hydroxycinnamic acid matrix and detected by positive-mode.