Marina Hincapie

A proteomics platform combining depletion, multi-lectin affinity chromatography (M-LAC), and isoelectric focusing to study the breast cancer proteome

The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies.

Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.

A two step fractionation approach for plasma proteomics using immunodepletion of abundant proteins and multi-lectin affinity chromatography: Application to the analysis of obesity, diabetes, and hypertension diseases

Plasma is an important biological material for biomarker discovery. However, the wide dynamic range in protein concentration remains a major challenge. In this paper, we introduce the development of a proteomic platform for analysis of plasma samples. The method utilizes a double fractionation approach which combines the MARS immunodepletion column with multi-lectin affinity chromatography, M-LAC, to deplete the most abundant proteins in plasma, the majority of which are glycosylated. To determine the suitability of this methodology, we applied the workflow described in this study to a sample set composed of four groups: a control pool and three different disease pools: obesity, diabetes, and hypertension. We were able to identify changes in the level of several proteins; for example, a protein such as angiotensinogen was found to be present at high levels in patients with obesity plus diabetes and hypertension. On the other hand, apolipoprotein CI was shown to be elevated in all disease groups. A review of the literature supported our observation. The methodology presented in this report was shown to be effective for profiling changes in the plasma proteome of subjects with obesity and its associated complications such as diabetes and hypertension.

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.

Proteomic Profiling of a High-Producing Chinese Hamster Ovary Cell Culture

The productivity of mammalian cell culture expression systems is critically important to the production of biopharmaceuticals. In this study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-X(L) gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomarker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene that is known to have growth regulating properties in human cells. A total of 392 proteins were identified in this study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were downregulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-X(L). The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures.

Using lectins to harvest the plasma/serum glycoproteome.

Aberrant protein glycosylation has been shown to be associated with disease processes and identification of disease‐specific glycoproteins and glycosylation changes may serve as potential diagnostic and therapeutic biomarkers. However despite recent advances in proteomic‐based biomarker discovery, this knowledge has not yet translated into an extensive mining of the glycoproteome for potential biomarkers. The major challenge for a comprehensive glycoproteomics analysis arises primarily from the enormous complexity and the large dynamic range in protein constituent in biological samples. Methods that specifically target glycoproteins are therefore necessary to facilitate their selective enrichment prior to their identification by MS‐based analysis. The use of lectins, with selective affinities for specific carbohydrate epitopes, to enrich glycoprotein fractions coupled with modern MS, have greatly enhanced the identification of the glycoproteome. On account of their ability to specifically bind cell surface carbohydrates lectins have, during the recent past, found extensive applications in elucidation of the architecture and dynamics of cell surface carbohydrates, glycoconjugate purification, and structural characterization. Combined with complementary depletion and MS technologies, lectin affinity chromatography is becoming the most widely employed method of choice for biomarker discovery in cancer and other diseases.

Towards an integrated proteomic and glycomic approach to finding cancer biomarkers

Advances in mass spectrometry have had a great impact on the field of proteomics. A major challenge of proteomic analysis has been the elucidation of glycan modifications of proteins in complex proteomes. Glycosylation is the most structurally elaborate and diverse type of protein post-translational modification and, because of this, proteomics and glycomics have largely developed independently. However, given that such a large proportion of proteins contain glycan modifications, and that these may be important for their function or may produce biologically relevant protein variation, a convergence of the fields of glycomics and proteomics would be highly desirable. Here we review the current status of glycoproteomic efforts, focusing on the identification of glycoproteins as cancer biomarkers.

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 (> or = 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.

Study of the human plasma proteome of rheumatoid arthritis

In this study, we report a combined proteomic and peptidomic analysis of human plasma from patients with rheumatoid arthritis (RA) and controls. We used molecular weight cut-off filters (MWCO: 10kDa) to enrich low-molecular-weight (LMW) peptides from human plasma. The peptide fraction was analyzed without trypsin digestion by capillary reversed-phase high-performance liquid chromatography (HPLC) coupled to a linear ion trap-FT-MS system, which identified 771 unique peptides in the peptidome study (from 145 protein progenitors). An anti-albumin/anti-IgG column was used to remove albumin and immunoglobulin G (IgG) from the human plasma. After that, the albumin/IgG-depleted sample was fractionated into a bound fraction and an unbound fraction on a multi-lectin affinity column (M-LAC). LC-MS analysis of the corresponding tryptic digests identified 308 proteins using the M-LAC approach. Relative differences in the following protein classifications were observed in the RA human plasma samples compared with controls: structural proteins, immuno-related proteins, protease inhibitors, coagulation proteins, transport proteins and apolipoproteins. While some of these proteins/peptides have been previously reported to be associated with RA disease such as calgranulin A, B, C and C-reactive protein, several others were newly identified (such as thymosin beta4, actin, tubulin, and vimentin), which may further the understanding of the disease pathogenesis. Moreover, we have found that the peptidomic and glycoproteomic approaches were complementary and allow a more complete picture of the human plasma proteome which can be valuable in studies of disease etiology.

The Development of an integrated platform to identify breast cancer glycoproteome changes in human serum

Protein glycosylation represents one of the major post-translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC-MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.