Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography

Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ∼7.0 femtomole detection limits and linear spectral response for proteins fractionated over ∼4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5–6× increase in the number of unique monoisotopic masses observed <30 kDa and an ∼4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate p<i>I</i> compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different p<i>I</i>. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues

Number of different probes between Copaxone, Glatopa, nonequivalent glatiramoid molecule ACN, and media (univariate analysis, <i>P</i> < 0.05^*).

<p>FDR, false discovery rate; FWER, familywise error rate.</p><p>*Student’s t-test.</p><p>Number of different probes between Copaxone, Glatopa, nonequivalent glatiramoid molecule ACN, and media (univariate analysis, <i>P</i> < 0.05^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#t001fn002" target="_blank">*</a>}).</p

MDS plots based on the 4176 Copaxone-responsive probes.

<p>As expected, clear separation is obtained between Copaxone and media groups. There is also clear separation between media and copolymer groups (ACN, Copaxone, or Glatopa), some separation between GA (Copaxone or Glatopa) and ACN and no visible separation between Copaxone and Glatopa. ACN, acetonitrile nonconforming copolymer; GA, glatiramer acetate; MDS, multidimensional scaling.</p

Methodology for the generation of murine GA-responsive Th2-polarized T cells.

<p>In vivo immunization of naive mice with Copaxone was followed by 13 rounds of ex vivo restimulation of the CD4⁺ T-cell population over 6 months for development of the Th2-455 line. APCs, antigen-presenting cells; GA, glatiramer acetate; IL-2, interleukin 2; Th, T-helper.</p

Gene expression levels in Th2-polarized cells exposed to Copaxone and Glatopa vs media alone.

<p>Th, T-helper.</p><p>*Log2 of the fold difference in mean expression for the indicated test material compared with media alone.</p><p>Gene expression levels in Th2-polarized cells exposed to Copaxone and Glatopa vs media alone.</p

Box plots for gene expression changes for key Th2 cytokines IL-4 and IL-3 and additional genes related to immune cell function.

<p>No statistically significant differences between Glatopa and Copaxone were observed for any of these genes.</p

T-helper cell pathway diagram.

<p>Transcripts measured in the current study are shown in the diagram as nodes and are colored based on the differences observed when cells are stimulated with Copaxone in comparison with cell culture media alone (<i>P</i> < 1e-3 [Student’s t-test]; red for increase, green for decrease). Molecules with <i>P</i> > 1e-3 are shown in gray. Genes refer to the human ortholog. Human HLA-DMA, HLA-DMB, and HLA-DQB1 represent murine H2-Dma, H2-Dmb2, and H2-Ab1, respectively. Blue arrows show the flow of activation by major Th1- and Th2-influencing molecules in the pathway. For example, APCs produce IL-4, which binds to and activates the IL-4 receptor, leading to the phosphorylation and activation of the transcription factor STAT6. Red and green arrows show the expected transcriptional outcomes of Th1 and Th2 polarization; red and green arrows indicate that activation will cause the transcript to increase and decrease, respectively. Expected transcriptional outcomes are based on reports in the literature on Th1/Th2 T-cell polarization and on studies conducted with GA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref003" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref004" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref008" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref010" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref029" target="_blank">29</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140299#pone.0140299.ref034" target="_blank">34</a>]. Gray lines indicate members of a group. APCs, antigen-presenting cells; CXCR1, chemokine (C-X-C motif) receptor 1; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR5, chemokine (C-X-C motif) receptor 5; CXCL10, chemokine (C-X-C motif) ligand 10; GA, glatiramer acetate; HLADMA, major histocompatibility complex class II, DM alpha; HLADMB, major histocompatibility complex class II, DM beta; HLADQB1, major histocompatibility complex class II, DQ beta 1; ICOSLG, inducible T-cell costimulatory ligand; IL-4, interleukin-4; MHC class II, major histocompatibility complex class II; LTB, lymphotoxin beta (tumor necrosis factor superfamily, member 3); MYD, myeloid differentiation primary response protein; S100A10, S100 calcium-binding protein A10; STAT, signal transducer and activator of transcription; TCR, T-cell receptor; Th, T-helper.</p

Number of significantly perturbed probes (q-value < 0.05 and fold change > 1.3) when comparing sample groups.

<p>An initial set of 4176 probes was detected by comparing Copaxone and media groups. This set yielded significant differences between Glatopa and media and between nonequivalent glatiramoid ACN and glatiramer acetate (Copaxone or Glatopa), but no significant difference between Copaxone and Glatopa.</p

Identification of critical residues of human (h)AQP4_281-300 for presentation in the context of <i>HLA-DRB1*03</i>:<i>01</i> and recognition by the B.10 T cell receptor (TCR).

<p>(A) First, the ability of hAQP4_281-300-reactive lymph node cells to recognize the alanine screening peptides was determined by ELISpot. 5.0x10⁵ cells/well lymph node cells taken ten days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with hAQP4_281-300 were restimulated with hAQP4 alanine scanning peptides (2 5 μg/mL) for 48 hours in IFNγ and IL-17 ELISpot plates (* = P-value < 0.05 and ** = P-value < 0.01). (B) Alanine screening peptides that not result in an increased frequency of IFNγ and IL-17 secreting lymph node cells were identified as the key residue peptides, and were subsequently tested in a MHC binding assay. Splenocytes taken from <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice were incubated for 12 hours in the presence of biotinylated hAQP4 alanine scanning peptides. Post incubation, cells were stained utilizing FITC-Avidin, and antigen positive cells were quantified by flow cytometry (* = P-value < 0.05 and ** = P-value < 0.01). (C) There was no Ig isotype class switch in mice immunized with mAQP4_284-299 with regard to antibody responses against whole-length AQP4 protein. (D) Critical <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, and B.10 TCR contact amino acids are specified. E₂₈₈ and L₂₉₄ are required as <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, while T₂₈₉, D₂₉₀, D₂₉₁, and I₂₉₃ are critical B.10 TCR interacting residues.</p

<i>HLA-DRB1*03</i>:<i>01</i> transgenic mice are disease resistant to active immunization with human aquaporin 4 (hAQP4), and adoptive transfer of hAQP4-specific T cells.

<p>(A) <i>HLA-DRB1*03</i>:<i>01</i> mice were actively immunized with proteolipid protein (PLP)_91-110 (100 μg/100 μl/mouse; positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152720#pone.0152720.ref025" target="_blank">25</a>]), or varying AQP4 antigens*(whole-length hAQP4 protein, hAQP4_281-300, murine (m)AQP4_281-300, hAQP4_281-300 with a Quil-A Incomplete Freund Adjuvant (IFA) booster on day 14 post-immunization, mAQP4_281-300 with a Quil-A IFA booster on day 14 post immunization, and hAQP4_281-300 plus mAQP4_281-300) emulsified in Complete Freund Adjuvant (CFA). Immunization with a positive control proteolipid protein (PLP)_91-110, a dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> led to typical EAE. (B) Lymph node cells taken from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 or mAQP4_281-300 were restimulated for three days and passively transferred into <i>HLA-DRB1*03</i>:<i>01</i> mice. None of these experimental approaches resulted in clinical disease. (C) Paraffin sections were stained with haematoxlin eosin (H&E) and luxol fast blue (LFB). Representative sections of the spinal cords from PLP_91-110 and hAQP4_281-300 immunized mice are shown. On histopathological examination there were no visible signs of cellular infiltration, inflammation, or demyelination within the brain and spinal cord in any experimental paradigms other than in active immunization with PLP_91-110, the dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> that led to typical EAE (spinal cord shown; inflammatory infiltrates and areas of demyelination are indicated by black arrows). (D) Fifteen days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with PLP_91-110 or hAQP4_281-300, pupillary reflex was measured via a mouse pupillometry. Mice actively immunized with hAQP4_281-300 and the control antigen PLP_91-110 did not show altered pupillary responses.</p