Ltq ion trap mass spectrometer

Separation of the (R)- and (S)- enantiomers of reticuline was performed using the chiral column CHIRALCEL OD-H (4.6x250mm, Daicel Chemical Industries) and the solvent system hexane:2-propanol:diethylamine (78:22:0.01) at a flow rate of 0.55 ml min^-1 [18 (link)]. Following LC separation, metabolites were injected into an LTQ ion trap mass spectrometer (Thermo Electron, San Jose, CA) and detected by selected reaction monitoring (SRM). SRM transitions of m/z 288→164.0 (CID@35) and 330→192 (CID@30) were used to detect reticuline. Retention times for reticuline obtained in samples matched retention times observed with authentic standards.

The mass spectrometer was cleaned, calibrated with the manufacturer’s standard mixture, tuned with GluFib and Angiotensin and tested for sensitivity prior to each replicate block by infusion of a dilution series of GluFib and Angiotensin. The mass accuracy and sensitivity of the LC–ESI–MS/MS system was tested with a tryptic digestion of a mixture of cytochrome C, glycogen phosphorylase B and alcohol dehydrogenase [26 (link)]. The plasma peptides were collected over a C18 preparative column with elution in 2 µl of 5% formic acid and 65% acetonitrile and immediately diluted with 18 µl of 5% formic acid for injections via a 20 µl loop with a Rheodyne manual injector. A total of ~ 5 µg of extracted and purified peptides was injected for each analytical HPLC separation over a 300 micron ID column (15 cm) with inline filter frits. The peptides were ionized at 4.5 kV via a micro electrospray ion source with 10 L N₂ per minute with a transfer capillary temperature of 200 °C into a Thermo Electron Corporation LTQ ion trap mass spectrometer [36 (link)]. The peptides were randomly and independently sampled by MS and MS/MS without replacement as the peptides eluted from the HPLC column into the electrospray source from 350 to 2000 m/z.

Liquid chromatography was performed on a Symmetry C18 column (3.5 µm, 50 × 2.1 mm; Waters, Milford, MA, USA), using a Surveyor HPLC system (Thermo Electron Corporation, Waltham, MA, USA). Separation was achieved using mobile phases A and B of H₂O and MeCN, respectively (both containing 2 mM ammonium formate and 0.01% formic acid). A linear gradient elution (0.25 mL/min) was used from 30 to 100% B over 20 min, held for 10 min, then returned to 30% B. The HPLC system was coupled to an LTQ ion trap mass spectrometer operating with an electrospray ionization (ESI) interface (Thermo Electron Corporation). Typical ESI parameters were spray voltage ±4.5 kV, heated capillary temperature 250 °C, and capillary at 35 or −20 V (in positive or negative modes). The mass spectrometer was operated in scan mode (m/z 400–1500). LC–MSⁿ experiments were conducted in positive ionization mode with collision energy (CE) 55 eV (1–6) and in negative ionization mode at CE 35 eV (1–3) or 40 eV (4–6). The mass range scanned was 10 units greater than the precursor m/z down to the lowest m/z value allowed by the ion trap mass spectrometer.

O-Linked oligosaccharides were released from PVDF membranes by reductive β-elimination as previously described [22 (link)] and analyzed by LC-ESI-MSⁿ using a 10 cm × 250 μm I.D. column, packed in house with 5 μm porous graphitized carbon particles (Thermo Scientific, Waltham, MA) and a linear gradient of 0 to 40% acetonitrile solution in water, containing 10 mM ammonium bicarbonate over 40 min with a static split flow rate of 250 μl/min bringing down the on-column flow rate to 5–10 μl/min.
Mass spectrometric data was collected in negative ion mode using a Thermo Scientific LTQ ion trap mass spectrometer (San Jose, CA) with an electrospray voltage of 3.5 kV, capillary voltage of −33.0 V, and capillary temperature of 300°C. The data were manually interpreted as described [23 (link)]. Structural assignment was performed by the comparison of MS² spectra from isolated chromatographic peaks to structures identified in the UniCarb-DB glycomic database [24 (link)].

One hundred microlitres (100 μl) of frozen MPs fractions were thawed and resuspended in 60% methanol and 0.1 M ammonium bicarbonate pH 8.0. Incubation with diothiothreitol followed by cysteine alkylation with iodoacetamide reduced the proteins. The solution was incubated overnight with mass spectrometry grade trypsin (Pierce, USA) at 37 °C to digest the proteins. The obtained tryptic peptides underwent desalting using reversed phase cartridge by washing in 0.5% acetic acid. The peptides were then eluted with 95% acetonitrile and 0.5% acetic acid. The eluted peptides were lyophilized and re-suspended in 0.5% acetic acid and directly analyzed using 2-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS). The samples were loaded onto a microcapillary strong cation exchange column and fractions were collected using an increasing salt step elution gradient. Afterwards, each fraction was analyzed using the LTQ ion trap mass spectrometer (Thermo-Fisher Scientific, Waltham, MA). The acquired MS/MS spectra were searched against protein database available at www.uniprot.org [19 (link)]. The protein interactions were acquired using the string database available at http://string-db.org/cgi/.