title: 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.
abstract: 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 (pI, hydrophobicity, and mass), 217 putative proteoforms were observed from 21 to 24 kDa and pI 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 pI 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.
authors.affiliation: Department of Pathology, University of Texas Southwestern Medical Center, TX, USA.
authors.forename: Junmei
authors.initials: J
authors.lastname: Zhang
authors.forename: John R
authors.initials: JR
authors.lastname: Corbett
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Plymire
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Greenberg
authors.forename: Steven M
authors.initials: SM
authors.lastname: Patrie
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000134:cerebrospinal fluid* / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D054834:Lipocalins / Q000134:cerebrospinal fluid* / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D020033:Protein Isoforms / Q000134:cerebrospinal fluid / Q000737:chemistry; D011499:Protein Processing, Post-Translational
chemical_list: D054834:Lipocalins; D020033:Protein Isoforms; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase
keywords: Biomedicine; Cyclooxygenase; Glycosylation; Neurodegeneration; Neuroinflammation