Multi-lectin Affinity Chromatography and Quantitative Proteomic Analysis Reveal Differential Glycoform Levels between Prostate Cancer and Benign Prostatic Hyperplasia Sera.

Currently prostate-specific antigen is used for prostate cancer (PCa) screening, however it lacks the necessary specificity for differentiating PCa from other diseases of the prostate such as benign prostatic hyperplasia (BPH), presenting a clinical need to distinguish these cases at the molecular level. Protein glycosylation plays an important role in a number of cellular processes involved in neoplastic progression and is aberrant in PCa. In this study, we systematically interrogate the alterations in the circulating levels of hundreds of serum proteins and their glycoforms in PCa and BPH samples using multi-lectin affinity chromatography and quantitative mass spectrometry-based proteomics. Specific lectins (AAL, PHA-L and PHA-E) were used to target and chromatographically separate core-fucosylated and highly-branched protein glycoforms for analysis, as differential expression of these glycan types have been previously associated with PCa. Global levels of CD5L, CFP, C8A, BST1, and C7 were significantly increased in the PCa samples. Notable glycoform-specific alterations between BPH and PCa were identified among proteins CD163, C4A, and ATRN in the PHA-L/E fraction and among C4BPB and AZGP1 glycoforms in the AAL fraction. Despite these modest differences, substantial similarities in glycoproteomic profiles were observed between PCa and BPH sera

Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins

We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.7 page(s

Automated platform for fractionation of human plasma glycoproteome in clinical proteomics

This publication describes the development of an automated platform for the study of the plasma glycoproteome. The method consists of targeted depletion in-line with glycoprotein fractionation. A key element of this platform is the enabling of high throughput sample processing in a manner that minimizes analytical bias in a clinical sample set. The system, named High Performance Multi-Lectin Affinity Chromatography (HP-MLAC), is composed of a serial configuration of depletion columns containing anti-albumin antibody and protein A with in-line multilectin affinity chromatography (M-LAC) which consists of three mixtures of lectins concanavalin A (ConA), jacalin (JAC), and wheat germ agglutinin (WGA). We have demonstrated that this platform gives high recoveries for the fractionation of the plasma proteome (≥ 95%) and excellent stability (over 200 runs). In addition, glycoproteomes isolated using the HP-MLAC platform were shown to be highly reproducible and glycan specific as demonstrated by rechromatography of selected fractions and proteomic analysis of the unbound (glycoproteome 1) and bound (glycoproteome 2) fractions.6 page(s

Kinetic analysis of glycoprotein-lectin interactions by label-free internal reflection ellipsometry

Introduction: Glycoproteomics is undergoing rapid development, largely as a result of advances in technologies for isolating glycoproteins and analyzing glycan structures. However, given the number and diversity of glycans, there is need for new technologies that can more rapidly provide differential carbohydrate–protein structural information on a large scale. We describe a new microarray platform based on a label-free imaging ellipsometry technique, which permits simultaneous detection of multiple glycoprotein–lectin interactions without the need for reporter labels, while still providing high throughput kinetic information at much lower cost. Our results demonstrate the utility of LFIRE™ (Label-Free Internal Reflection Ellipsometry) for the rapid kinetic screening of carbohydrate–lectin recognition. The technology was also used to evaluate the benefits of the lectin immobilization format using multi-lectin affinity chromatography (M-LAC) to capture glycoproteins (with enhanced binding strength or avidity) from biological samples. Using a printed panel of lectins, singly or in combination, we examined the binding characteristics of standard glycoproteins. Results and Discussion: Using kinetic measurements, it was observed that the binding strength of lectins to carbohydrates is enhanced using a multi-lectin strategy, suggesting that improved selectivity and specificity can lead to increased functional avidity. The data presented confirm that this label-free technology can be used to effectively screen single or combinations of lectins. Furthermore, the combination of LFIRE™ and M-LAC may permit more rapid and sensitive identification of novel biomarkers based on carbohydrate changes in glycoproteins, and lead to a better understanding of the connections of glycan function in cellular mechanisms of health and disease.10 page(s

Additional file 1: Table S1. of Assessing biological and technological variability in protein levels measured in pre-diagnostic plasma samples of women with breast cancer

Subject characteristics of blood plasma samples from the Northern California site of the Breast Cancer Family Registry. Table S2 Targeted proteins in the antibody-based proteomics platforms. (DOCX 25 kb

Additional file 2: of Assessing biological and technological variability in protein levels measured in pre-diagnostic plasma samples of women with breast cancer

Measured Protein Levels Across LC-MS/MS, Olink, and Myriad-RBM platforms. (XLSX 98 kb

LARP1 binding to hepatitis C virus particles is correlated with intracellular retention of viral infectivity

Hepatitis C virus (HCV) virions contain a subset of host liver cells proteome often composed of interesting virus-interacting factors. A proteomic analysis performed on double gradient-purified clinical HCV highlighted the translation regulator LARP1 on these virions. This finding was validated using post-virion capture and immunoelectron microscopy, as well as immunoprecipitation applied to in vitro (Huh7.5 liver cells) grown (Gt2a, JFH1 strain) and patient-derived (Gt1a) HCV particles. Upon HCV infection of Huh7.5 cells, we observed a drastic transfer of LARP1 to lipid droplets, inducing colocalization with core proteins. RNAi-mediated depletion of LARP1 using the C911 control approach decreased extracellular infectivity of HCV Gt1a (H77), Gt2a (JFH1), and Gt3a (S52 chimeric strain), yet increased their intracellular infectivity. This latter effect was unrelated to changes in the hepatocyte secretory pathway, as evidenced using a functional RUSH assay. These results indicate that LARP1 binds to HCV, an event associated with retention of intracellular infectivity

FIG4 is a hepatitis C virus particle-bound protein implicated in virion morphogenesis and infectivity with cholesteryl ester modulation potential

There is growing evidence that virus particles also contain host cell proteins, which provide viruses with certain properties required for entry and release. A proteomic analysis performed on double-gradient-purified hepatitis C virus (HCV) from two highly viraemic patients identified the phosphatidylinositol 3,5-bisphosphate 5-phosphatase FIG4 (KIAA0274) as part of the viral particles. We validated the association using immunoelectron microscopy, immunoprecipitation and neutralization assays in vitro as well as patient-derived virus particles. RNA interference-mediated reduction of FIG4 expression decreased cholesteryl ester (CE) levels along with intra- and extracellular viral infectivity without affecting HCV RNA levels. Likewise, overexpressing FIG4 increased intracellular CE levels as well as intra- and extracellular viral infectivity without affecting viral RNA levels. Triglyceride levels and lipid droplet (LD) parameters remained unaffected. The 3,5-bisphosphate 5-phosphatase active site of FIG4 was found to strongly condition these results. Whilst FIG4 was found to localize to areas corresponding to viral assembly sites, at the immediate vicinity of LDs in calnexin-positive and HCV core-positive regions, no implication of FIG4 in the secretory pathway of the hepatocytes could be found using either FIG4-null mice, in vitro morphometry or functional assays of the ERGIC/Golgi compartments. This indicates that FIG4-dependent modulation of HCV infectivity is unrelated to alterations in the functionality of the secretory pathway. As a result of the documented implication of CE in the composition and infectivity of HCV particles, these results suggest that FIG4 binds to HCV and modulates particle formation in a CE-related manner

A HUPO test sample study reveals common problems in mass spectrometry-based proteomics

We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.8 page(s