On-membrane digestion of beta-casein for determination of phosphorylation sites by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry.
This article discusses the features of a newly developed matrix-assisted laser desorption/ionization quadrupole/time-of-flight (MALDI-QqTOF) mass spectrometer that is useful in the analysis of phosphorylated peptides. Aliquots of beta-casein, a commonly used phosphorylated protein standard, were digested with trypsin directly on a non-porous polyurethane membrane used as sample support in MALDI-QqTOF mass spectrometry (MS) experiments. Although a complete peptide map was obtained, it was difficult to obtain sequence information for some of the tryptic fragments, in particular T1-2, which bears four phosphate groups and is thus difficult to ionize in positive mode. This article focuses on the sequencing of this particular fragment by comparing MS/MS spectra obtained using different precursor ions. These precursors associated with T1-2 were [M + H](+), [M + H](2+), and [M + H - nH(3)PO(4)](+) ions. Typically, phosphorylated ions showed facile unimolecular losses of phosphoric acid moieties, and produced limited backbone fragmentation. The abundance of [M + H](2+) ions of T1-2 in the full mass spectrum was low relative to that of [M + H](+). [M + H - 4H(3)PO(4)](+) ions as MS/MS precursors underwent backbone fragmentations, with phosphoserine residues transformed into dehydroalanines or serines. Unusual b + 18 u fragments were observed, although only for segments with previously phosphorylated serines. These partly interfered with c-ions, and were noticeable due to overlapping isotopic envelopes. It was possible to establish the sequence of phosphorylated tryptic fragment T1-2 and the location of phosphate groups using the mass of dehydroalanine residues (69 Da) and b + 18 u fragments as markers. All MS and MS/MS spectra obtained with fully phosphorylated beta-casein were compared with spectra acquired with dephosphorylated beta-casein obtained commercially. These comparisons helped assess the spectral differences caused by the presence of phosphate groups. Also, they highlighted the potential usefulness of conducting dephosphorylation directly on the probe prior to MALDI analysis in future studies.
Lee
C H
CH
University of Manitoba, Department of Chemistry, Winnipeg, MB R3T 2N2, Canada.
McComb
M E
ME
Bromirski
M
M
Jilkine
A
A
Ens
W
W
Standing
K G
KG
Perreault
H
H
D002364:Caseins / Q000032:analysis*; D008567:Membranes, Artificial; D010710:Phosphates / Q000032:analysis; D010766:Phosphorylation; D011140:Polyurethanes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods
D002364:Caseins; D008567:Membranes, Artificial; D010710:Phosphates; D011140:Polyurethanes