Ltq orbitrap mass spectrometer

In order to evaluate the enrichment capability of the MSP@PNAMAm magnetic hybrid microspheres, we further used MSP@PNAMAm to enrich glycopeptides in human serum from normal volunteers. The tryptic digest of 5 μL human serum was treated according to the above procedure after reduction and alkylation. Then the eluate was collected and sent for nano-LC-MS/MS analysis. The human serum protein sample was analyzed by a LC-20AD system (Shimadzu, Tokyo, Japan) connected to a LTQ orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, CA, USA). The lyophilized deglycosylated peptides were redissolved in solution containing 5% ACN containing 0.1% FA. Then the sample solution was loaded on a CAPTRAP column (0.5 mm × 2 mm, MICHROM Bioresources, Auburn, CA, USA) in 4 min with a flow rate of 20 μL/min. For a gradient separation, the gradient elution was performed as follows: Acetonitrile from 5% to 45% (95% ACN in 1% FA) over 100 min at a flow rate of 500 nL/min. For each cycle of duty, full mass scan was acquired from 400 to 2000 m/z. The MS/MS spectra were obtained in data-dependent ddMS2 mode. The 12 most intense ions with charge 2, 3 or 4 were selected for the MS/MS run, and the a dynamic exclusion duration was 90 s. Finally, three parallel enrichment operations were performed as technical repeats.

Peptides were re-dissolved in 20 μl 0.1% acetic acid and analyzed by an LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA, USA) equipped with an HPLC system (Eksigent, Redwood City, CA). Samples were trapped on a 5 mm Pepmap 100 C18 (Dionex, Sunnyvale, CA) column (300 μm ID, 5 μm particle size) and then analyzed on a 200 mm Alltima C18 homemade column (100 μm ID, 3 μm particle size). Separation was achieved by using a mobile phase consisting of 5% acetonitrile, 94.9% H₂O, 0.1% acetic acid (solvent A) and 95% acetonitrile, 4.9% H₂O, 0.1% acetic acid (solvent B), with a linear gradient from 5 to 40% solvent B in 40 min at a flow rate of 400 nl/min. Eluted peptides were electro-sprayed into the LTQ-Orbitrap operated in a data-dependent mode. Mass spectrometric data was searched against the Uniprot proteomics database (version 2013-01-06) with MaxQuant software (version 1.3.0.5) to obtain peptides and proteins identified in each experiment, as well as their label-free abundance. Search parameters were: MS accuracy 6 ppm, MS-MS accuracy 0.5 Da, fixed modification of cysteine alkylation with acrylamide, variable modification of methionine oxidation and protein N-terminal acetylation, digestion with trypsin, protein hits containing at least one unique peptide, and false discovery rates of both peptides and proteins within 0.01.

Affinity-purified complexes were sequentially digested with LysC peptidase and trypsin. LC–MS/MS was carried out by nanoflow reversed phase liquid chromatography (RPLC) (Eksigent, CA) coupled on-line to a Linear Ion Trap (LTQ)-Orbitrap mass spectrometer (Thermo-Electron Corp). A cycle of full FT scan mass spectrum (m/z 350–1800, resolution of 60,000 at m/z 400) was followed by 10 MS/MS spectra acquired in the LTQ with normalized collision energy (setting of 35%). Following automated data extraction, resultant peak lists for each LC–MS/MS experiment were submitted to Protein Prospector (UCSF) for database searching^{46 (link)}. Each project was searched against a normal form concatenated with the random form of the T. brucei database (http://tritrypdb.org/tritrypdb/).

After lyophilization, the most active fraction was dissolved in 0.1% formic acid and 2% acetonitrile for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a LTQ-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany). Samples were loaded onto a reversed-phase C18 column with a diameter of 0.1 mm × 10 cm and a particle size of 3 μm. Separation of the peptides was achieved by using a gradient of 5 to 35% acetonitrile in 0.1% formic acid over 120 min. The acquired MS/MS data were transformed to MGF by BIWORKS, and the data were searched against the NCBI database using MASCOAT software.
To investigate antihypertensive activity on SHRs, antioxidant capacity and mode of inhibition of ACE, the purified peptide was synthesized by Shanghai Bootech BioScience & Technology Co., Ltd. The purity of the synthetic peptide was 99% as evidenced by HPLC analysis. The IC₅₀ value of the peptide was investigated according to the method described above.

Furin cleavage reactions were set up as described above, except that BSA was not added to the reactions. The reaction mixtures were incubated for 60 min at room temperature and were stopped by adding EGTA to 10 mM concentration to inhibit furin. Reactions where 10 mM EGTA was present at the time of furin addition were carried out for controls. The peptides arising from furin cleavage were purified using C18 StageTips. The peptides were separated using Agilent 1200 series nanoflow system (Agilent Technologies) and sprayed into an LTQ Orbitrap mass spectrometer (Thermo Electron) with a nanoelectrospray ion source (Proxeon). The data was analyzed using MaxQuant^{49 (link)}.

To investigate USP53-binding proteins in hBMSCs, the cells were transfected with lentiviral control vector or lentiviral vector-USP53 and cultured in osteogenic induction medium for 4 days. The cells were then treated with MG132 (10 μM) for 6 h, and hBMSCs were harvested using passive lysis buffer (Promega). Total protein (approximately 500 μg) was incubated with an antibody against GFP (cat. no. sc-390394; Santa Cruz Biotechnology) and IgG control (cat. no. 12-370; Milipore) at 4 °C overnight. SDS-PAGE was performed, following which the gels were stained with an InstantBlue staining kit (cat. no. ISB1L; Sigma). Differential bands were collected for LC-MS/MS, which was performed using a LTQ orbitrap mass spectrometer (Thermo Electron, San Jose, CA, USA).