The resulting
peptide mixtures
were analyzed by reversed-phase chromatography using a Waters nanoAcquity
UPLC pump coupled online to an LTQ/Orbitrap mass spectrometer. Peptides
were loaded onto a Symmetry 180 μm × 2 cm C18 trap column,
which was washed and placed in-line with a Waters BEH130 C18 analytical
column (75 μm × 25 cm). As the peptides elute off the column
they are ionized into the gas phase of the mass spectrometer. The
peptide ion masses are measured with resolution of 60 000 at m/z 400 by the Orbitrap, followed by MS/MS
fragmentation of the six most intense precursors with charge state
≥2 above an intensity threshold of 10 000 in the LTQ
ion trap. Dynamic exclusion was used to avoid repeat sequencing, with
a repeat count of one and exclusion duration of 30 s for precursors
within 10 ppm. The MS/MS spectra were extracted and searched against
the human Uniprot (release 2013_01) using the MASCOT search engine,
version 2.2 (Matrix Science) with a mass tolerance on precursor ions
of 25 ppm and 0.5 Da for fragment ions. The following variable modifications
were considered: carbamidomethyl-Cys, methionine oxidation, and pyro-glutamic
acid for N-terminal Gln. Peptide identifications were accepted at
a 1% FDR threshold based on a reversed protein database search. Protein
assembly and label-free quantification by spectral counting was performed
using Isoform Resolver.51 (link)
peptide mixtures
were analyzed by reversed-phase chromatography using a Waters nanoAcquity
UPLC pump coupled online to an LTQ/Orbitrap mass spectrometer. Peptides
were loaded onto a Symmetry 180 μm × 2 cm C18 trap column,
which was washed and placed in-line with a Waters BEH130 C18 analytical
column (75 μm × 25 cm). As the peptides elute off the column
they are ionized into the gas phase of the mass spectrometer. The
peptide ion masses are measured with resolution of 60 000 at m/z 400 by the Orbitrap, followed by MS/MS
fragmentation of the six most intense precursors with charge state
≥2 above an intensity threshold of 10 000 in the LTQ
ion trap. Dynamic exclusion was used to avoid repeat sequencing, with
a repeat count of one and exclusion duration of 30 s for precursors
within 10 ppm. The MS/MS spectra were extracted and searched against
the human Uniprot (release 2013_01) using the MASCOT search engine,
version 2.2 (Matrix Science) with a mass tolerance on precursor ions
of 25 ppm and 0.5 Da for fragment ions. The following variable modifications
were considered: carbamidomethyl-Cys, methionine oxidation, and pyro-glutamic
acid for N-terminal Gln. Peptide identifications were accepted at
a 1% FDR threshold based on a reversed protein database search. Protein
assembly and label-free quantification by spectral counting was performed
using Isoform Resolver.51 (link)
