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MALDI-TOF : Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles. -
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Abstract
Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.
Keywords: Glycosylation, Concanavalin A, boronic acid, magnetic particles, MALDI-TOF MS
Protein glycosylation and glycoproteomics are growing fields of interest due to the relationship between glycosylation degree and type and the health status of cells. The discovery and identification of glycosylated peptides and proteins and the analyses of their glycostructures are increasingly important in diagnosis and proteomics. In particular, missing, aberrant, or additional glycosylations are known to be linked to certain diseases and may be utilized as markers for diagnosis and/or therapeutic monitoring.1 Direct mass-spectrometric detection of glycosylated peptides and proteins from complex sources such as blood serum/plasma, urine, and spinal fluid is quite difficult due to saline contaminants disturbing the MS ionization process. Furthermore, highly abundant proteins (human albumin in blood serum) interfere with the mass spectrometric analysis. Therefore, enrichment and purification are essential prior to MALDI-TOF MS analysis and the subsequent identification and structure elucidation. The diagnostic applications require an automated workflow to assure ease of handling, good reproducibility, rapid analysis time, and high-throughput capability. The use of MALDI-TOF mass spectrometry and magnetic particles for identifying peptides and proteins and their corresponding modifications ideally fulfills those requirements.
Different approaches for the specific isolation of glycosylated peptides and proteins have been described, e.g., lectin affinity chromatography or boronic acid chromatography. Concanavalin A (ConA) is a lectin that specifically binds mannosyl and glucosyl residues containing unmodified hydroxyl groups at positions C3, C4, and C6.2,3 It can be utilized for the targeted binding of certain oligosaccharide structures of N-glycosylated proteins.4 The pentacore of high mannose, hybrid, or bi-antennary complex–type oligosaccharide structures is shown in Figure 11.. In contrast, phenyl boronic acid covalently binds molecules containing cis-diol groups.5 Glycopeptides and glycoproteins equipped with saccharides like mannose, galactose, or glucose can form heterocyclic diesters that are stable under alkaline conditions (Figure 22).). Additionally, boronic acid facilitates the enrichment of the more heterogeneous O-linked oligosaccharides.
MATERIALS AND METHODS
Model proteins and chemicals were purchased from Sigma-Aldrich, Deisenhofen, Germany. Carboxyl- and boronic acid–functionalized magnetic particles were obtained from Chemicell, Berlin, Germany.
Carboxyl-functionalized magnetic beads were covalently modified with ConA employing the carbodiimide method according to the protocol from Dynal Biotech, Oslo, Norway, with a few modifications. The resulting beads are available as MB-LAC-ConA Kit (magnetic bead-lectin affinity chromatography with Concanavalin A) including all buffers from Bruker Daltonik, Leipzig, Germany.
Binding and Elution of Proteins
Binding of samples on MB-LAC ConA beads was performed according to the manufacturer’s recommendations. Briefly, beads and sample were incubated for 1 h at RT under gentle agitation in binding buffer. The beads were separated from the supernatant using a magnetic separation device (Bruker Daltonik, Leipzig, Germany). After washing, the bound glycoproteins/peptides were eluted under acidic conditions.
Boronic acid–functionalized beads were loaded with glycoprotein containing sample under slightly alkaline (pH 8.5) conditions. After incubation for 1 h at RT under gentle shaking, the bound protein species were eluted under acidic conditions.
Mass Spectrometry
For MS analysis the eluted samples were applied onto Anchor-Chip 600 targets (Bruker Daltonik, Germany) using 2,5-dihydroxyacetophenone (2,5-DHAP) (Bruker Daltonik, Germany) as matrix according to the manufacturer’s recommendations. MS measurements were performed on an autoflex II TOF/TOF. Spectra were acquired in the linear mode.
SDS-PAGE
For SDS-PAGE, samples were mixed with 5X loading buffer, boiled for 5 min at 95°C, and applied onto a discontinuous 12.5% or 15% poly-acrylamid gel according to the protocol of Laemmli.6 After running, the gels were stained with Coomassie blue.
RESULTS AND DISCUSSION
Enrichment of Glycoproteins by ConA Beads
Carboxyl-functionalized magnetic particles were employed to couple ConA onto the beads using the carbodiimide method. The quality and the specificity of the MB-LAC-ConA beads were demonstrated by the capturing of different model proteins, i.e., bovine serum albumin (BSA), bovine lactoferrin, ovalbumin (OVA), bovine RNase B, and bovine transferrin (1–5 μg, corresponding to 10–300 pmol of protein). BSA served as non-glycosylated negative control. All other proteins contain N-linked oligosaccharide structures of different N-glycan types. Binding of proteins on MB-LAC-ConA beads (20 μL) was performed under neutral conditions for 1 h at RT. After binding, washing, and elution, the supernatants and the eluates were analyzed by MALDI-TOF mass spectrometry (Figure 3A3A)) and SDS-PAGE (Figure 3B3B).). Preparations for MALDI-TOF MS were performed with 2,5-DHAP on Anchor-ChipTM 600 targets, and spectra were acquired on an autoflex II TOF/TOF.