Targeting serum glycoprotein cancer biomarkers with click chemistry Courtenay Hart1, Brian Agnew1, Tamara Nyberg1, Mahbod Hajivandi2, Marshall Pope2 and Rajkumar Lakshmanaswamy3 1Molecular Probes-Invitrogen, 29851 Willow Creek Road, Eugene, Oregon 97402 USA; 2Invitrogen Corporation, 1600 Faraday Ave., Carlsbad, California 92008 USA; 3Texas Tech University Health Sciences Center at El Paso, 4800 Alberta Ave, El Paso, Texas 79905 Differential 2D gel analysis of Click-iT™ labeled whole serum from Her2/neu vs. normal mice Introduction Extensive changes to both O-linked and N-linked sugars may include: Glycoproteins have been historically difficult to study • • • • Vehicle sialidase 5 Ac4GalNAz O HN AcO AcO OAc N3 AcO OAc O OAc 2 5 3 1 3 2 6 1 8 7 1 TAMRA SYPRO Alkyne Ruby Protein Stain 7 TAMRA Alkyne SYPRO® Ruby Glycoproteins Protein Gel Stain Click-labeled protein samples ZOOM® IEF Fractionator TAMRA Alkyne SYPRO® Ruby Glycoproteins Protein Gel Stain Con Gal Man Con Gal Man Con Gal Man Con Gal Man N H N H N H N H N H N H N H N H N H N H N H N H TAMRA Alkyne pH 4 pH 4.6-5.4 GlcNAc Total O-glycoproteins GalNAc Gal Man NeuNAc pH 6.2-7.0 TAMRA Alkyne Nuclear and cytosolic Normal Mouse O-GlcNAc glycoproteins Normal Mouse Her2Neu Mouse pH 4.5 pH 5.5 pH 4.5 pH 5.5 pH 4.5 pH 5.5 pH 4.5 pH 5.5 pH 5.3 pH 6.3 pH 5.3 pH 6.3 pH 5.3 pH 6.3 pH 5.3 pH 6.3 N N N The copper(I)-catalyzed azide-alkyne cycloaddition reaction forms an irreversibly labeled fluorescent protein (via triazole linkage). pH 6.1 pH 7.1 pH 6.1 pH 7.1 pH 6.1 pH 7.1 pH 6.1 pH 7.1 pH 6 pH 10 pH 6 pH 10 pH 6 pH 10 pH 6 pH 10 • Sacrifice mice, prepare serum extracts. • Remove albumin from serum samples using a Q-Proteome column (Qiagen). • Sialidase digestion: Incubate albumin-depleted serum in 0.25 U/mL Sialidase A (Prozyme) at 4 °C, pH 6 for 2 hours. • Label with TAMRA-alkyne using the Click-iTTM TAMRA Glycoprotein Detection Kit (Invitrogen). • IEF fractionation: Resolubilize 2 mg proteins in 7 M urea, 2 M thiourea, 65 mM DTT, 2% CHAPS, 2% ASB-14, 50 mM DTT. Separate into five pH fractions using the Zoom IEF fractionator (Invitrogen). • 1D PAGE: Separate 10 μg samples on 4-12% Bis-Tris gels with MOPS buffer. • 2D PAGE: Reduce and alkylate proteins, precipitate, resolubilize in 7 M urea, 2 M thiourea, 65 mM DTT, 2% CHAPS, 2% ASB-14, and 0.5% carrier ampholytes. Separate 50 μg unfractionated samples on pH 4-7 IEF strips and 100 μg IEF fractionated samples on respective narrow pH range IEF strips. Separate all samples in the second dimension on 4-12% Bis-Tris gels with MOPS buffer. SYPRO® Ruby Protein Stain Terminal GlcNAc residues in pH 4 pH 7 untreated Her2/neu or normal 4 mouse serum were labeled with 5 GalNAz using GalT (Y289L) enzyme and then click-labeled No ID with TAMRA alkyne dye. Proteins of interest were excised from the same gels after poststaining with SYPRO Ruby protein gel stain (red circles). pH 7 pH 4 pH 7 4 Protein IDs are shown in Table 5 1. A band identified as IgG heavy chain by Protein-G enrichment is heavily labeled in No ID Her2/neu mouse serum in comparison to normal mouse serum (blue star). Unfortunately, we were not able pH 7 pH 4 pH 7 to confirm the identity by MS fingerprinting. Also of interest is that GalT specifically labels only the higher molecular weight isoform of transferrin, which corresponds to the asialo b2 isoform, but not the b1 isoform. pH 7 • Azido sugar incorporation was selective, as demonstrated by enzymatic (sialidase) treatment. • Click labeling of glycoproteins is compatible with mass spectrometric analyses. • GalT (Y289L) enzymatic labeling of terminal GlcNAc residues resulted in the labeling of numerous proteins, some of which were differentially modified or expressed. • The click labeling method combined with IEF fractionation allows for the identification of low abundance serum glycoproteins. References GalNAz IP injection of azido-modified sugar (3 days, GlcNAz, 1.2 gram/kg in DMSO or 7 days, ManNAz and GalNAz, 300 mg/kg in DMSO). N Fluorescently-tagged GlcNAc-modified protein Metabolic azido sugar labeling of Her2/neu and normal mice combined with click chemistry detection resulted in the efficient labeling of a unique subset of serum glycoproteins, allowing for the identification of differentially modified glycoproteins. Experimental Methods • N • GalNAz C u (I), R T, 30 min N 3 GalNAz Click-iT™ Detection of Glycoproteins3,4 N Fluorescent Alkyne Probe Results and Conclusions SYPRO® Ruby Protein Gel Stain Her2Neu Mouse GalNAz Cell surface sialylated glycoproteins pH 7.0-10.0 N3 H2N Control pH 4 N3 N3Azide-tagged GlcNAc GalT (Y289L) enzymatic labeling of Her2/neu versus normal mouse serum Run 1-D or narrow pH range 2-D gels N3 + Azide-tagged GlcNAc pH 4 Red boxes indicate GalNAz labeled fractions that were further analyzed on narrow pH range 2D gels. -N 3 UDP-GalNAz Cu(I), RT, 30 min N3 IP inject axidomodified sugars into Her2/neu or normal mouse N3 SYPRO Ruby Protein Stain 66 4 ° C, ON GlcNAc-modified protein O N3 TAMRA Alkyne β-GalT1 (Y289L) + Enzyme N3 NH O _+ _ + 3 Differential 1D and 2D gel analysis of Click-iT™ labeled IEF-fractionated serum from Her2/neu (H) vs. normal (N) mice showing enrichment of low abundance proteins OAc OAc AcO AcO _ + _ + Enzymatic labeling of GlcNAc-modified proteins in activated Her2/neu and normal mice with GalT (Y289L) enzyme5,6 Ac4GlcNAz OAc O OAc NH O-Glycoproteins (GalNAz) Sialic Acids (ManNAz) 1 6 8 In vivo metabolic labeling of glycoproteins in activated Her2/neu or normal mice1,2 Ac4ManNAz 3 Normal GlcNAz • pH 7 Blue circles depict examples of TAMRA labeled proteins that show differential labeling. Red circles depict areas on the same gels that were excised after post-staining with SYPRO® Ruby protein gel stain. Proteins IDs are shown in Table 1. A band identified as IgG heavy chain by Protein-G enrichment is present in high amounts in Her2/neu mouse serum compared to normal mouse serum and is labeled by GlcNAz (blue star). We were unable to ID with MALDI analysis. Glycan structures are highly heterogeneous. Analytical methods require harsh chemical conditions. Lectins have overlapping selectivities and low affinities. Radioactive labeling is expensive and dangerous. Selective glycan antibodies are difficult to produce. Glycoproteins/peptides are not HPLC or MS friendly. • pH 4 H2N GlcNAz • Her2Neu Mouse pH 7 pH 3-4.6 • pH 4 pH 5.4-6.2 • Normal Mouse pH 7 unfractionated • Increased glycan branching Altered sialylation patterns Altered fucosylation patterns Truncated glycan chains Altered O-mucin secretion pH 4 ManNAz • SYPRO® Ruby Protein Gel Stain Her2Neu Mouse pH 7 GlcNAz Altered glycosylation patterns are a hallmark of malignant transformation TAMRA Alkyne Normal Mouse pH 4 GalNAz Altered glycosylation patterns are a hallmark of malignant transformation. Both N- and O-linked glycoproteins display abnormal glycosylation patterns in malignant tumor cells and in the serum of cancer patients. Here we describe a rapid, highly sensitive, fluorescence-based in-gel detection method for the identification of serum glycoprotein biomarkers using a mouse cancer model. The approach utilizes in vivo metabolic labeling combined with click chemistry detection. Analyses of blood serum glycoproteins from Her2/neu mice bearing multiple breast tumors is compared with normal mice of the same species. ManNAz is specifically incorporated into sialic acids as demonstrated by sialidase sensitivity Table 1. Protein identification by MALDI peptide fingerprinting Sample No. Protein ID Swiss Prot Accession Number 1. Jacobs CL, Yarema KJ, Mahal LK, Nauman DA, Charters NW, Bertozzi CR; (2000) Methods Enzymol. 327:260-275. 2. 3. 4. Dube DH and Bertozzi CR; (2003) Curr. Opin. Chem. Biol. 7(5) 616-625. Kolb HC, Finn MG, Sharpless KB; (2001) Angew. Chem. In. Ed. V40:2004-2021. Tornøe CW, Christensen C, Meldal M; (2002) J. Org. Chem. May 3; 67(9):3057-64. 5. Khidekel N, Arndt S, Lamarre-Vincent N, Lippert A, Poulin-Kirstien KG, Ramakrishnan B, Qasba PK, Hsieh-Wilson LC; (2003) J. Am. Chem. Soc. Dec 31; 125(52):16162-3. 6. Boeggeman EE, Ramakrishnan B, Qasba PK; (2003) Protein Expr. Purif. Aug 30; (2):21929. NCBI Accession Number Mass pI Sequence Coverage 1 Alpha-1-antitrypsin 1-6 P81105 gi|68068019 43335 5.25 24% 2 Albumin P07724 gi|33859506 65892 5.53 28% 3 Hemopexin Q91X72 gi|18044757 48960 7.59 21% 4 Transferrin Q921I1 gi|18606172 74881 6.81 28% 5 Alpha-1-B-glycoprotein isoform 2 Q19L12 gi|94399515 56554 6.33 30% 6 Liver carboxylesterase N P23953 gi|2921308 59182 5.06 15% • Image TAMRA signal on a FX Pro Plus scanner (Bio-Rad) using 532 nm excitation and 555 nm long-pass emission. • Stain gels with SYPRO® Ruby Gel Stain (Invitrogen), image on a FX Pro Plus scanner using 488 nm excitation and 555 nm long-pass emission. 7 Alpha-2-macroglobulin 35 kDa subunit Q61838 gi|2492496 28225 7.09 41% • Mass spectrometry: Excise proteins from gel, trypsin digest and analyze by MALDI/TOF on a 4700 Protein Analyzer (ABI). 8 Serine proteinase inhibitor A3K (Contrapsin) P07759 gi|15079234 44742 5.05 42%
© Copyright 2024 ExpyDoc
