Finnigan ltq mass spectrometer

Nano LC connected to Finnigan LTQ mass spectrometer (Thermo Scientific, Waltham MA) was used. Biphasic columns were prepared in-house for analysis; the columns contained 365 μm OD (outer diameter) × 100 μm ID (inside diameter) fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA). The capillaries were packed using a pressure cell with helium and 9 cm of C18-AQ 5 μm reverse phase (PP), followed by 3 cm of 5 μm strong cation exchange Luna resin. The desalted sample was loaded onto the in-house column in 20 μL of a mixture of 5% acetonitrile and 0.1% formic acid.
Reversed-phase chromatography was performed using a binary buffer system of 0.1% formic acid (buffer A) and acetonitrile in 0.1% formic acid (buffer B). Nano LC was performed by using a linear gradient of 3–50% of buffer B at a flow rate of 0.200 μL/min. The peptides were eluted in the course of an 11-step program of increasing concentration of salt solution. The eluent was ionized by electrospraying from the column directly into the MS/MS system. A parent-ion scan was performed in the range of 400–1600 m/z (mass-to-charge ratio). The top five most intense parent ions were chosen, and an MS/MS-ion scan was performed by collusion-induced dissociation. The run time for LC-MS/MS was 120 min for each step. The total run time for the 11 steps was approximately 22 h. A total of 7 MudPIT runs were conducted.

LC-ESI-MS/MS analyses were performed on a Finnigan LTQ mass spectrometer (Thermo Scientific) connected to an Acquity ultraperformance liquid chromatography (UPLC) system (Waters Corporation) as described previously (56 (link)), except for the UPLC conditions: Buffer A contained 10 mM NH₄CH₃CO₂, 2% CH₃CN, 1% CH₃OH, and 97% H₂O (v/v), pH 7.0, and buffer B contained 10 mM NH₄CH₃CO₂, 95% CH₃CN, 1% CH₃OH, and 4% H₂O (v/v), pH 7.0 (Table 2, Figs. 8 and S14–S18; Tables S4–S9). ESI settings: spray voltage 4.5 kV, sheath gas flow 40, auxiliary gas flow rate 15, sweep gas flow rate 5, capillary voltage −49 V, tube lens voltage −140 V, and capillary temperature 270 °C.
The fully extended products were identified (Table 2, Figs. 8 and S14–S18; Tables S4–S9) by comparing the observed CID fragments with the theoretical values using a Mongo Oligo Mass Calculator v2.06 (http://rna.rega.kuleuven.be/masspec/mongo.htm). The relative yields of extended products were calculated based on the peak areas of extracted ion chromatograms.