Diagnostic Potential of Transferrin Glycoforms—A Lectin‐Based Protein Microarray Approach

Abstract

Purpose Disease or a specific condition may cause alteration of human transferrin (hTf) glycosylation pattern. A specific analytical platform, lectin‐based protein microarray, is designed and optimized for the investigation of hTf glycans, attached to the protein core in their native form. Experimental design hTf molecules isolated from healthy persons of different age, diabetes mellitus type 2 (T2DM) or colorectal carcinoma (CRC) patients are used for method validation. Reliability of the results is ensured by three criteria for the evaluation of hTf–lectin interactions: i) signal‐to‐noise ratio above 3, ii) signal intensity above 250 arbitrary units, and iii) hTf concentration ensuring high sensitivity of the assay. Results Six lectins, out of fourteen tested, satisfy the criteria. hTf is spotted at concentration of 50 µg mL−L. When physiological samples (isolated hTf) are analyzed, the highest potential to differentiate between population groups expresses Aleuria aurantia (AAL), Triticum vulgaris (WGA) and Phaseolus vulgaris (PHA‐E) lectins. The initial amount of hTf which can be analyzed is very low (75 pg). Conclusion and clinical relevance Results confirm that a very sensitive, high‐throughput lectin‐based protein microarray platform can be formulated to detect changes in hTf glycan structures which can be considered as biomarkers of ageing or a disease.

Glycosylation Biomarkers Associated with Age-Related Diseases and Current Methods for Glycan Analysis

“…For instance, significant changes in high-mannose and fucosylated biantennary complex n -glycans have been observed in the serum of prostate cancer patients [ 86 ], as shown in Table 3 . Similarly, preliminary data states that there are pronounced age-associated changes at the level of fucosylation and branching of glycans in transferrin, as well as an increased degree of fucosylation in colorectal carcinoma [ 82 ]. These findings are consistent with previous studies in which colorectal cancer patients had dramatically higher levels of sialylation and fucosylation [ 83 ].…”

“…Hence, pre-treatment of the sample is required to successfully characterise the full glycome. Regarding the determination techniques, a broad range of analytical platforms can be applied for the profiling, characterisation and analysis of glycans, being the most widely used liquid chromatography coupled with fluorescence detection (LC-FLR) [ 9 , 48 , 97 , 100 ], lectin-based microarray [ 50 , 82 , 85 ], capillary electrophoresis (CE) [ 58 , 71 , 118 ], matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) [ 52 , 75 , 92 , 112 ], LC-MS [ 23 , 24 , 49 , 96 ], DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) [ 19 , 22 , 74 , 79 ], ion mobility (IM) and NMR [ 93 ]. Several reviews focus exclusively on describing the available techniques used for glycan analysis [ 129 , 130 , 131 ].…”

“…Lectin-based protein microarray is a method that enables the determination of released n -glycans in their native form. It has been reported that a very sensitive lectin-based protein microarray assay can be formulated and used to detect changes in glycan structures which accompany ageing or a disease [ 82 ]. Lectin-based microarrays are optimal for the determination of glycosylation changes and fits very well with biomarker research requirements [ 143 ], replacing conventional lectin-based analytical methods such as lectin blotting [ 85 ].…”

Identification of Whole-Serum Glycobiomarkers for Colorectal Carcinoma Using Reverse-Phase Lectin Microarray

“…Thus, the SNA I lectin should be a positive discriminator when used for the age matrix (i.e., hY vs. hO), as was experimentally confirmed in this study ( Figure 1A ). In the other study using the reverse-phase lectin microarray, it was found that the SNA I binding to transferrin isolated from human serum decreased with age ( 40 ), suggesting that there might be other protein carriers besides IgG-carrying glycans recognized by SNA I.…”

Non-Enzymatic Glycation of Transferrin and Diabetes Mellitus

“…37 Penezić et al showed that the structure of serum Tf glycans differed between the different healthy people with varied ages and type 2 diabetes or colon cancer patients, as assessed by lectin-based protein chip technology. 38 Compared to healthy controls, oral squamous cell carcinoma patients have different expression levels of N-glycotypes of several serum proteins, such as Tf, IgG1, IgG4, and haptoglobin. 39 The serum profile of Tf isoforms is altered in patients with rheumatoid arthritis (RA) and RA patients treated with rituximab.…”