Computational protocol: The Haemophilus influenzae HMW1C Protein Is a Glycosyltransferase That Transfers Hexose Residues to Asparagine Sites in the HMW1 Adhesin

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Protocol publication

[…] The complex mixtures of peptides and glycopeptides from HMW1802–1406 were analyzed using high-resolution nano-LC-MS on a hybrid mass spectrometer consisting of a linear quadrupole ion-trap and an Orbitrap (LTQ-Orbitrap XL, Thermo-Fisher). The liquid chromatographs were nanoflow HPLC systems (NanoLC-1Dplus™ and NanoLC-Ultra™) that were interfaced to the mass spectrometer with a nanospray source (PicoView PV550; New Objective). The in-house packed LC column (Jupiter C12 Proteo, 4 µm particle size, 90 Å pore size [Phenomenex]) was equilibrated in 98% solvent A (aqueous 0.1% formic acid) and 2% solvent B (acetonitrile containing 0.1% formic acid). The samples (10 µL) were injected from autosampler vials using the LC-systems autosamplers at a flow rate of 1.0 µL/min and were eluted using a segmented linear gradient (250 nL/min) with solvent B: isocratic at 2% B, 0–2 min; 2% B to 40% B, 2–65 min; 40% B to 80% B, 65–70 min; isocratic at 80% B, 70–72 min; 80% B to 2% B, 72–77 min; and isocratic at 2% B, 77–82 min. The survey scans (m/z 350–2000) (MS1) were acquired at high resolution (60,000 at m/z = 400) in the Orbitrap, and the MS/MS spectra (MS2) were acquired in the linear ion trap at low resolution, both in profile mode. The maximum injection times for the MS1 scan in the Orbitrap and the LTQ were 50 ms and 100 ms, respectively. The automatic gain control targets for the Orbitrap and the LTQ were 2×105 and 3×104, respectively. The MS1 scans were followed by six MS2 events in the linear ion trap with wideband collision activation in the ion trap (parent threshold = 1000; isolation width = 2.0 Da; normalized collision energy  = 30%; activation Q = 0.250; activation time = 30 ms). Dynamic exclusion was used to remove selected precursor ions (−0.25/+1.5 Da) after MS2 acquisition with a repeat count of 2, a repeat duration of 30 s, and a maximum exclusion list size of 200. The following ion source parameters were used: capillary temperature 200 °C, source voltage 2.5 kV, source current 100 µA, and the tube lens at 79 V. The data were acquired using Xcalibur, version 2.0.7 (Thermo-Fisher).The MS2 spectra were analyzed both by searching a customized protein database that contained the sequences of HMW802–1406 and by expert manual interpretation. The exact masses of the glycopeptides and fragmentation ions were calculated using the Molecular Weight Calculator, version 6.45 (http://ncrr.pnl.gov/software/). For database searches, the LC-MS files were processed using MASCOT Distiller (Matrix Science, version 2.3.0.0) with the settings previously described . The resulting MS2 centroided files were used for database searching with MASCOT, version 2.1.6, and the following parameters: enzyme, trypsin; MS tolerance = 10 ppm; MS/MS tolerance = 0.8 Da with a fixed carbamidomethylation of Cys residues and the following variable modifications: Methionine, oxidation; Pyro-glu (N-term); Maximum Missed Cleavages  = 5; and 1+, 2+, and 3+ charge states. […]

Pipeline specifications

Software tools Molecular Weight Calculator, Mascot Distiller
Application MS-based untargeted proteomics
Organisms Haemophilus influenzae
Chemicals Galactose, Glucose