Plasma proteome plus site‐specific N‐glycoprofiling for hepatobiliary carcinomas

Abstract

Hepatobiliary cancer is the third leading cause of cancer death worldwide. Appropriate markers for early diagnosis, monitoring of disease progression, and prediction of postsurgical outcome are still lacking. As the majority of circulating N ‐glycoproteins are originated from the hepatobiliary system, we sought to explore new markers by assessing the dynamics of N ‐glycoproteome in plasma samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), or combined HCC and CCA (cHCC‐CCA). Using a mass spectrometry‐based quantitative proteomic approach, we found that 57 of 5358 identified plasma proteins were differentially expressed in hepatobiliary cancers. The levels of four essential proteins, including complement C3 and apolipoprotein C‐III in HCC, galectin‐3‐binding protein in CCA, and 72 kDa inositol polyphosphate 5‐phosphatase in cHCC‐CCA, were highly correlated with tumor stage, tumor grade, recurrence‐free survival, and overall survival. Postproteomic site‐specific N ‐glycan analyses showed that human complement C3 bears high‐mannose and hybrid glycoforms rather than complex glycoforms at Asn85. The abundance of complement C3 with mannose‐5 or mannose‐6 glycoform at Asn85 was associated with HCC tumor grade. Furthermore, stepwise Cox regression analyses revealed that HCC patients with a hybrid glycoform at Asn85 of complement C3 had a lower postsurgery tumor recurrence rate or mortality rate than those with a low amount of complement C3 protein. In conclusion, our data show that particular plasma N ‐glycoproteins with specific N ‐glycan compositions could be potential noninvasive markers to evaluate oncological status and prognosis of hepatobiliary cancers.

Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma

“…We adopted emPAI% using a fixed amount of protein for each sample in spite of the fact that absolute protein levels have yet to be known to perform an LC-MS/MS-based intersample, comparative proteomic study. Our previous study enrolled patients with HCC, CCA, or combined hepatocellular-cholangiocarcinoma, in which a protein biomarker pool containing 57 entities to discriminate the patients from the controls was established and the clinical relevance of different glycosylation patterns on complement C3 in HCC was subsequently analyzed [6]. Although using the same dataset, we used a different strategy here to identify specific plasma biomarkers for HCC or CCA.…”

“…The proteins were then cleaned and concentrated using Amicon Ultra-0.5 mL centrifugal filters (molecular weight cutoff: 3,000 Da) device (Merck Millipore, Darmstadt, Germany) and digested at 37˚C overnight using sequencing grade trypsin (Promega, Fitchburg, WI, USA) in 10 mM ammonium bicarbonate in an enzyme-to-substrate ratio of 1:50. We followed the procedure of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis outlined in our previous study [6]. A rapid separation liquid chromatography system (Ultimate 3000; Dionex, Santa Clara, CA, USA) equipped with a C18 column (Acclaim PepMap RSLC, 75μm × 150 mm, 2 μm, 100 Å) was coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific).…”

Recent Advances of Functional Proteomics in Gastrointestinal Cancers- a Path towards the Identification of Candidate Diagnostic, Prognostic, and Therapeutic Molecular Biomarkers

“…They identified several glycoproteins with elevated N-glycosylation levels correlated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3) and galectin-3-binding protein (LGALS3BP), which were involved in several well-known pancreatic cancer pathways such as: TGF-β, TNF, NF-kappa-B and TFEB related lysosomal changes [ 123 ]. The majority of serum/plasma N-glycoproteins are produced in the hepatobiliary system and an unhealthy situation in the liver, such as HCC results in aberrant serum/plasma N-glycome [ 11 ], therefore exploring these alterations in HCC patients is highly valuable in order to identify potential biomarkers. Chang and colleagues investigated the dynamics of N-glycoproteome in plasma samples from patients with HCC, cholangiocarcinoma (CCA), or combined HCC and CCA (cHCC-CCA) to find potential biomarkers related to disease diagnosis and progression; totally 57 differentially expressed proteins were identified, of those, C3 and APOC3 proteins were associated with the tumor stage, tumor grade, recurrence-free survival and overall survival of HCC.…”

“…Moreover, C3 bearing Man5 or hybrid glycoforms were associated with post-surgery prognosis of HCC even stronger than the whole level of complement C3. In addition, complement C3 with hybrid glycoforms were related with the mortality rate of HCC [ 11 ]. In another study, Zhang and colleagues, in order to identify potential serum N-linked glycoproteins related to HCC recurrence, used lectin affinity chromatography combined with enzyme-catalyzed 18 O 3 − or 16 O 3 − labeling and screened serum samples from 100 early-stage HCC patients; results were validated using immunohistochemical staining in 193 HCC tissues.…”

“…Several proteomics studies focusing on PTMs in GI cancers have been published so far; including protein phosphorylation [ 10 ], glycosylation [ 11 ], acetylation [ 12 ], methylation [ 13 ] and ubiquitination [ 14 ]. Protein phosphorylation and glycosylation in particular have been extensively studied in GI cancers because of their key role in regulating a wide range of cellular signaling pathways associated with initiation, promotion and progression of the disease.…”