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Proteoform analysis of lipocalin‐type prostaglandin D ‐synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry
Author(s) -
Zhang Junmei,
Corbett John R.,
Plymire Daniel A.,
Greenberg Benjamin M.,
Patrie Steven M.
Publication year - 2014
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201300368
Subject(s) - lipocalin , chemistry , glycan , cerebrospinal fluid , acetylation , mass spectrometry , isoelectric focusing , prostaglandin , chromatography , biochemistry , glycoprotein , biology , enzyme , gene , neuroscience
Lipocalin‐type prostaglandin D ‐synthase (L‐PGDS) in cerebrospinal fluid contributes to the maturation and maintenance of the CNS. L‐PGDS PTMs may contribute to pathobiology of different CNS diseases, but methods to monitor its proteoforms are limited. Herein, we combined off‐gel IEF and superficially porous LC (SPLC) with Fourier transform MS to characterize common cerebrospinal fluid L‐PGDS proteoforms. Across 3D physiochemical space (p I , hydrophobicity, and mass), 217 putative proteoforms were observed from 21 to 24 kDa and p I 5–10. Glycoprotein accurate mass information, combined with MS/MS analysis of peptides generated from 2D‐fractionated proteoforms, enabled the putative assignment of 208 proteoforms with varied PTM positional occupants. Fifteen structurally related N ‐glycans at N29 and N56 were observed, with different N ‐glycan compositional variants being preferred on each amino acid. We also observed that sialic acid content was a major factor for p I shifts between L‐PGDS proteoforms. Other putative PTMs characterized include a core‐1 HexHexNAc‐O‐glycan at S7, acetylation at K16 and K138, sulfonation at S41 and T142, and dioxidation at C43 and C145. The IEF‐SPLC‐MS platform presented provides 30–40× improved peak capacity versus conventional 2DE and shows potential for repeatable proteoform analysis of surrogate PTM‐based biomarkers.