Relative Quantitation of Glycoisoforms of Intact Apolipoprotein C3 in Human Plasma by Liquid Chromatography–High-Resolution Mass Spectrometry

Glycosylation is one of the most important post-translational modifications to mammalian proteins. Distribution of different glycoisoforms of certain proteins may reflect disease conditions and, therefore, can potentially be utilized as biomarkers. Apolipoprotein C3 (ApoC3) is one of the many plasma glycoproteins extensively studied for association with disease states. ApoC3 exists in three main glycoisoforms, including ApoC3-1 and ApoC3-2, which contain an O-linked carbohydrate moiety consisting of three and four monosaccharide residues, respectively, and ApoC3-0 that lacks the entire glycosylation chain. Changes in the ratio of different glycoisoforms of ApoC3 have been observed in pathological conditions such as kidney disease, liver disease, and diabetes. They may provide important information for diagnosis, prognosis, and evaluation of therapeutic response for metabolic conditions. In this current work, a liquid chromatography­(LC)–high-resolution (HR) time-of-flight (TOF) mass spectrometry (MS) method was developed for relative quantitation of different glycoisoforms of intact ApoC3 in human plasma. The samples were processed using a solid-phase extraction (SPE) method and then subjected to LC–full scan HRMS analysis. Isotope peaks for each targeted glycoisoform at two charge states were extracted using a window of 50 mDa and integrated into a chromatographic peak. The peak area ratios of ApoC3-1/ApoC3-0 and ApoC3-2/ApoC3-0 were calculated and evaluated for assay performance. The results indicated that the ratio can be determined with excellent reproducibility in multiple subjects. It has also been observed that the ratios remained constant in plasma exposed to room temperature, freeze–thaw cycles, and long-term frozen storage. The method was applied in preliminary biomarker research of diabetes by analyzing plasma samples collected from normal, prediabetic, and diabetic subjects. Significant differences were revealed in the ApoC3-1/ApoC3-0 ratio and in the ApoC3-2/ApoC3-0 ratio among the three groups. The workflow of intact protein analysis using full scan HRMS established in this current work can be potentially extended to relative quantitation of other glycosylated proteins. To our best knowledge, this is the first time that a systematic approach of relative quantitation of targeted intact protein glycoisoforms using LC–MS has been established and utilized in biomarker research

NF-κB activity in the presence of NleH variants.

<p>HEK293T cells were co-transfected with a luciferase reporter plasmid under the control of consensus κB sites, a β-galactosidase plasmid and a control (pCMV), NleH or OspG vector. After 40 hours, cells were stimulated by the addition of TNF-α (25 ng/ml; 24 hours). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033408#s2" target="_blank">Results</a> represent three biological replicates, where variants were tested in triplicate and assayed in duplicate. Statistical analysis with one-way ANOVA shows no significant difference compared with the pCMV control. Error bars represent the standard error of the mean.</p

Expression of NleH-GFP and Tir-GFP in <i>E. coli</i> O157:H7 defined LEE regulator mutants.

<p><i>E. coli</i> O157:H7 ZAP193, ZAP193Δ<i>ler</i> and ZAP193Δ<i>grlA</i> were transformed with constructs expressing NleH1-GFP (pAHE8; A), NleH2-GFP (pAHE22; B) and Tir-GFP (pAJR132; C). GFP expression was monitored during growth of the transformants in MEM media, with a promoterless GFP construct (pAJR70) as a background control. Fluorescence values were corrected for background and lines represent the average of three biological repeats.</p

Expression of NleH-GFP constructs in <i>E. coli</i> O157:H7 grown in defined media.

<p>Constructs consisting of 120 bp (pAHE18), 283 bp (pAHE19) or 531 bp (pAHE8) of the NleH1 5′ UTR and 113 bp (pAHE20), 291 bp (pAHE21) or 655 bp (pAHE22) of the NleH2 5′ UTR cloned upstream of <i>gfp</i> were transformed into ZAP193, grown in MEM-HEPES (A) or DMEM (B) and GFP fluorescence measured during growth. All values were corrected for background from a promoter-less GFP (pAJR70) control measured at the same optical density. Graphs represent the average of three experimental repeats.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Fluorescence microscopy of NleH-GFP.

<p>pAHE8 (NleH1-GFP) and pAHE22 (NleH2-GFP) were transformed in ZAP193, ZAP193Δler and ZAP193ΔgrlA and at OD₆₀₀ = 0.8, dried onto a microscope slide in 4% PFA and stained for EspA filaments. Volocity quantification software was used to determine the average GFP fluorescence per voxel of 100 individual bacteria for NleH1 (A) and NleH2 (B). Each point represents the average GFP fluorescence from a composite from 16 z-slice images thus reducing planar effects. Error bars represent the standard deviation.</p

Expression of NleH-GFP upon <i>E. coli</i> O157:H7 ZAP193 contact with EBL cells.

<p>ZAP193 transformed with plasmids expressing GFP constitutively (pAJR145; <i>rpsm</i>::<i>gfp</i>) or translational fusions of <i>nleH</i> or <i>tir</i> to <i>gfp</i> under the control of their native promoter (pAHE8; NleH1-GFP, pAHE22; NleH2-GFP, pAJR75; Tir-GFP) were added to EBL cells and incubated for 0, 5, 30, 60 or 180 minutes at 37°C, 5% CO₂ before the removal of supernatant and fixation of cells with 4% paraformaldehyde. The panel of images is representative of all time points tested, apart from Tir-GFP, that showed strong early expression during cell contact but was markedly reduced at 180 minutes.</p

Quantitative PCR of NleH transcripts in LEE regulator knockouts.

<p>RNA was collected from ZAP193 strains WT, Δler and ΔgrlA grown to OD₆₀₀ = 1.2 in MEM and cDNA prepared. NleH1, NleH2, GapA, Tir and 16S RNA transcript was then quantified by q-PCR, NleH values normalised to that of 16S RNA, and the fold change calculated comparing mutant to wild-type. Bars represent the average of three biological samples. Error bars represent the standard error of the mean.</p

Identification of YhaO and YhaJ as potential virulence determinants.

<p>(A) Screening of the <i>yhaOMKJ</i> locus for a role in virulence. SDS-PAGE profile of secreted proteins from TUV93-0, <i>yhaO</i>, <i>yhaM</i>, <i>yhaK</i> and <i>yhaJ</i> cultured in MEM-HEPES. Arrows indicate the location of the major LEE-encoded secreted effectors Tir, EspD and EspA as identified by mass-spectrometry. Samples were normalized according to cellular OD⁶⁰⁰ to normalize loading into each well. Immunoblot analysis of EspD levels from secreted (Sec) and whole cell lysate (WCL) fractions confirmed the SDS-PAGE results. Anti-GroEL was used to verify equal concentrations of WCL, which corresponded to OD⁶⁰⁰ normalized culture samples, loaded into each well (B) SDS-PAGE analysis highlighting complementation of the <i>ΔyhaO</i> and <i>ΔyhaJ</i> phenotypes by plasmids p<i>yhaO</i> and p<i>yhaJ</i>. SDS PAGE and immunoblot analysis of secreted protein profiles and EspD cytoplasmic expression confirmed the results. Protein secretion experiments were performed on multiple occasions.</p

Genomic and phylogenomic context of the <i>yhaOMKJ</i> locus.

<p>(A) Genomic context of the D-serine tolerance locus (blue) in three distinct <i>E</i>. <i>coli</i> isolates–CFT073 (UPEC), EDL933 (EHEC) and MG1655 (K-12). The system encodes DsdC (a LysR type transcriptional regulator), DsdX (a D-serine outer membrane transporter) and DsdA (a D-serine deaminase). In EDL933 the D-serine tolerance locus is truncated and replaced with the sucrose utilization locus (<i>cscRAKB</i> highlighted in green). (B) Genomic context of the second putative D-serine sensory locus (red) in CFT073, EDL933 and MG1655. The system encodes YhaJ (a putative LysR type transcriptional regulator), YhaK (a redox-sensitive bicupin), YhaM (a putative deaminase) and YhaO (a putative inner membrane D-serine transporter). (B) The <i>yhaOMKJ</i> locus is highly conserved across the <i>E</i>. <i>coli</i> phylogeny. Circularized phylogenomic tree of 1591 <i>E</i>. <i>coli</i> and <i>Shigella</i> isolates overlaid with gene carriage for the <i>dsdCXA</i> locus and the <i>yhaOMKJ</i> locus. The <i>yhaOMKJ</i> genes are indicated by red blocks and the <i>dsdCXA</i> locus by blue blocks. Ordering of the genes is numbered and corresponds to the gene in the legend labeled *. Presence of a gene is determined by > 80% identity over > 80% of the coding sequence. Pseudogenes are indicated as yellow blocks. <i>E</i>. <i>coli</i> phylogroups are subdivided by color with the branch point labeled on the tree. Phylogroup A = Blue; Phylogroup B1 = Green; Phylogroup B2 = Red; Phylogroup C = Magenta; Phylogroup D = Purple; Phylogroup E = Cyan; Phylogroup F = Brown; <i>Shigella</i> = Gold. The position of prototypical strains is indicated on the outside of the figure.</p