C18 nanoacquity beh analytical column

One-microgram aliquots of tryptic peptides per sample (injection volume 5 μL) were analyzed on a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled to a 5600 Triple TOF mass spectrometer (AB SCIEX) with a nanoelectrospray ion source. Samples were injected into a C18 PepMap trap column (5 µm, 100 µm I.D. × 2 cm, Thermo Scientific) at 2 µL/min, in 0.1% formic acid in water, and the trap column was connected on-line to a C18 nanoAcquity BEH analytical column (1.7 µm, 100 Å, 75 µm I.D. × 15 cm, Waters). The nanopump provided a flow-rate of 250nL/min and the gradient elution conditions were as follows: 0.1% formic acid in water as mobile phase A, and 0.1% formic acid in acetonitrile as mobile phase B, from 5 to 40% B in 120 min. The mass spectrometer operated in data-dependent acquisition mode. For MS1 scans, the accumulation time was set to 250 ms and up to 10 precursor ions were acquired per spectrum (100 ms for each MS2), which represents a total cycle time of 1.3 s. This shorter duty cycle allows for the acquisition of a greater number of points per MS1 ion precursor, which is essential to improve quantitation in label-free based quantitative approaches.

Peptide fractions were subjected to LC-MS/MS analysis using a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus) coupled to a high speed Triple TOF 5600 mass spectrometer (SciEx) with a nanoelectrospray ion source. Samples were injected on a C18 PepMap trap column (5 μm, 100 μm I.D. x 2 cm; Thermo Scientific) at 2 μL/min, in 0.1% formic acid in water, and the trap column was switched on-line to a C18 nanoAcquity BEH analytical column (1.7 μm, 100 Å, 75 μm I.D. x 15 cm, Waters), equilibrated in mobile phase A (0.1% formic acid in water), and peptide was eluted in a 120 min linear gradient from 5–40% B (0.1% formic acid in acetonitrile) at 250 nL/min. The mass spectrometer was operated in data-dependent acquisition mode. For TOF scans, accumulation time was set to 250 ms, and up to 15 precursor ions were monitored per cycle.

Samples of digested PG (muropeptides) were analysed on a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled to a 5600 Triple TOF mass spectrometer (AB SCIEX, Foster City, CA) with a nano-electrospray ion source. Samples were injected on a C18 PepMap trap column (5 μm, 100 μm I.D. x 2 cm, Thermo Scientific) at 2 μL/min, in 0.1% formic acid in water, and the trap column was switched on-line to a C18 nanoAcquity BEH analytical column (1.7 μm, 100 Å, 75 μm I.D. x15 cm, Waters). Equilibration was done in mobile phase A (0.1% formic acid in water), and peptide elution was achieved in a 40 min linear gradient from 5% - 40% B (0.1% formic acid in acetonitrile) at 250 nL/min. Instrument calibration was continuously updated through automatic injection and calibration of a commercial peptide mixture (Beta-gal tryptic digest, AB SCIEX). Mass accuracy ranged between 5–10 ppm. The mass spectrometer operated in data-dependent acquisition mode. For TOF scans, the accumulation time was set to 250 ms, and per cycle, up to 15 precursor ions were monitored. MS1 and MS2 spectra were analysed manually using PeakView software (SCIEX).