O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Abstract

INTRODUCTION: Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers. MATERIALS AND METHODS: In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent (18)O/(16)O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to (18)O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results. CONCLUSION: This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.

Fig. 1

Strategy used for the enrichment of glycosylated proteins for ¹⁶O/¹⁸O labeling based quantitative mass spectrometric analysis of liver proteins in HCC. Tissue lysates (4 mg) were prepared from tumor and non-tumor liver tissues from a diagnosed case of HCC and incubated with mixture of lectins. The lectin-bound proteins were eluted using mixture of sugars and bound proteins derived from tumor and non-tumor samples were resolved separated on SDS-PAGE. In-gel trypsin digestion was carried out in the presence of ¹⁶O/¹⁸O-water differential labeling of peptides derived from non-tumor/tumor samples. After mixing the labeled and unlabeled peptides, the LC-MS/MS analysis was done using quadrupole time-of-flight mass spectrometer

Fig. 2

Differential proteomic analysis by ¹⁸O labeling: Proteins identified only in lectin affinity enrichment. Panel of MS spectra and MS/MS spectra showing fold changes and peptide sequence identification from overexpressed proteins in HCC. Panels A, B, and C show the MS and MS/MS spectra of HTLNQIDEVK (fetuin), YALYDAS-FETK (destrin) and ADLSGITGAR (SERPINA1), respectively. The fold changes after lectin affinity enrichment is indicated in MS spectra

Fig. 3

Quantitative analysis of protein changes by whole proteome and lectin enrichment analysis. Panels A and B show the MS and MS/MS spectra of GGYTLVSGYPK (hemopexin) and TVDNFVALATGEK (peptidylprolyl isomerase B), respectively. The fold changes at the whole proteome level and after lectin affinity enrichment are indicated in MS spectra

Fig. 4

Quantitative analysis of protein changes by whole proteome and lectin enrichment analysis. Panel A and B show the MS and MS/MS spectra of TLMALGSLAVTK (transgelin) and YALYDATYETK (cofilin 1), respectively. The fold changes at the whole proteome level and after lectin affinity enrichment are indicated in MS spectra

Fig. 5

Quantitative analysis of protein changes by whole proteome and lectin enrichment analysis. Panels A and B show the MS and MS/MS spectra of GLESTTLADK (CSRP 1) and LSILYPATTGR (perox-iredoxin 6), respectively. The fold changes at the whole proteome level and after lectin affinity enrichment are indicated in MS spectra

Fig. 6

Quantitative analysis of protein changes by whole proteome and lectin enrichment analysis. Panels A and B show the MS and MS/MS spectra of ETFTTGLDAPR (tenascin C) and QLSSGVSEIR (HSP 27-kDa), respectively. The fold changes at the whole proteome level and after lectin affinity enrichment are indicated in MS spectra

Fig. 7

Validation of fetuin and cysteine-rich protein by Western blotting and immunohistochemical labeling in tissues used for proteomic analysis. A The homogenates from the tissue samples were resolved by SDS-PAGE and subsequently electroblotted onto nitrocellulose membrane and probed with specific antibodies as indicated. B Shows immunohistochemical labeling for fetuin and CSRP1, respectively

Fig. 8

Immunohistochemical labeling for alpha-2HS-glycoprotein proteins in tissue microarrays. Six representative sections from tissue microarrays (TMAs) containing 114 tumor and 79 normal tissues are shown

Fig. 9

MS/MS spectra of peptides from fibrinogen-like 2 and complement component 4A analyzed using QToF. The spectra A and B show the N-glycosylation sites of the peptide LDGST*NFTR from fibrinogen-like 2 and GL*NVTLSSTGR from complement component 4A respectively (where *N indicates the N-glycosylation site modified by ¹⁸O). Spectrum shows the conversion of Asn to Asp after PNGase F treatment

Fig. 10

Validation of candidate proteins by Western blotting in serum. Serum samples (7 mg) from HCC patients and normal pooled sample was enriched for glycoproteins using mixture of lectins as explained in methods. Eluted proteins were resolved by SDS-PAGE and subsequently electroblotted onto nitrocellulose membrane and probed with antibodies for destrin and transgelin