Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.
ABSTRACT: Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.
Project description:Glycosylation is an important post-translational modification of proteins. Many diseases, such as cancer, have proved to be related to aberrant glycosylation. High throughput quantitative methods have gained attention recently in the study of glycomics. With the development of high-resolution mass spectrometry, the sensitivity of detection in glycomics has largely improved; however, most of the commonly used MS-based techniques are focused on relative quantitative analysis, which can hardly provide direct comparative glycomic quantitation results. In this study, we developed a novel multiplex glycomic analysis method on an LC-ESI-MS platform. Reduced glycans were stable isotopic labeled during the permethylation procedure, with the use of iodomethane reagents CH2DI, CHD2I, CD3I, 13CH3I, 13CH2DI, 13CHD2I, 13CD3I, and CH3I. Up to 8-plex glycomic profiling was possible in a single analysis by LC-MS, and a 100 k mass resolution was sufficient to allow a baseline resolution of the mass differences among the 8-plex labeled glycans. The major advantages of this method are that it overcomes quantitative fluctuations caused by nanoESI, it facilitates a level of comparative quantitative glycomic analysis that accurately reflects the quantitative information in samples, and it dramatically shortens analysis time. Quantitation validation was tested on glycans released from bovine fetuin and model glycoprotein mixtures (RNase B, bovine fetuin, and IgG) with good linearity (R2 = 0.9884) and a dynamic range from 0.1 to 10. The 8-plex strategy was successfully applied to a comparative glycomic study of cancer cell lines. The results demonstrate that different distributions of sialylated glycans are related to the metastatic properties of cell lines and provide important clues for a better understanding of breast cancer brain metastasis.
Project description:Recently developed carbonyl-reactive aminoxy tandem mass tag (aminoxyTMT) reagents enable multiplexed characterization and quantitative comparison of structurally complex glycans between different biological samples. Compared to some previously reported isotopic labeling strategies for glycans, the use of the aminoxyTMT method features a simple labeling procedure, excellent labeling efficiency, and reduced spectral complexity at the MS(1) level. Presence of the tertiary amine functionality in the reporter region of the aminoxyTMT labels leads to increased ionization efficiency of the labeled glycans thus improving electrospray ionization (ESI)-mass spectrometry (MS) detection sensitivity. The use of the labeling reagent also makes electrophoretic separation of the labeled neutral and acidic glycans feasible. In this work, we characterized the ESI and collision induced dissociation (CID) behavior of the aminoxyTMT-labeled neutral and sialylated glycans. For the high-mannose N-glycans and small sialylated oligosaccharides, CID fragmentation of [M + Na + H](2+) provides the most informative MS(2) spectra for both quantitative and qualitative analysis. For complex N-glycans, MS(3) of the protonated Y1(H) ion can be used for relative quantification without interference from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides (HMOs) and different types of N-glycans released from glycoprotein standards were demonstrated. Improved resolution and quantification accuracy of the labeled HMO isomers was achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from human serum protein digests were labeled with six-plex aminoxyTMT and subjected to CE-ESI-MS/pseudo-MS(3) analysis, which demonstrated the potential utility of this glycan relative quantification platform for more complex biological samples.
Project description:Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-Glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.
Project description:Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.
Project description:For decades, the association between aberrant glycosylation and many types of cancers has been shown. However, defining the changes of glycan structures has not been demonstrated until recently. This has been facilitated by the major advances in MS and separation science, which allows the detailed characterization of glycan changes associated with cancer. MS glycomics methods have been successfully employed to compare the glycomic profiles of different human specimens collected from disease-free individuals and patients with cancer. Additionally, comparing the glycomic profiles of glycoproteins purified from specimen collected from disease-free individuals and patients with cancer has also been performed. These types of glycan analyses employing MS or LC-MS allow the characterization of native, labeled and permethylated glycans. This review discusses the different glycomic and glycoproteomic methods employed for defining glycans as cancer biomarkers of different organs, including breast, colon, esophagus, liver, lung, ovarian, pancreas and prostate.
Project description:Mass spectrometry based comparative glycomics is essential for disease biomarker discovery. However, developing a reliable quantification method is still a challenging task.We here report an isotopic labeling strategy employing stable isotopic iodomethane for comparative glycomic profiling by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). N-Glycans released from model glycoproteins and blood serum samples were permethylated with iodomethane ('light') and iodomethane-d1 or -d3 ('heavy') reagents. Permethylated samples were then mixed at equal volumes prior to LC/ESI-MS analysis.Peak intensity ratios of N-glycans isotopically permethylated (Heavy/Light, H/L) were almost equal to the theoretical values. Observed differences were mainly related to the purity of 'heavy' iodomethane reagents (iodomethane-d1 or -d3). The data suggested the efficacy of this strategy to simultaneously quantify N-glycans derived from biological samples representing different cohorts. Accordingly, this strategy is effective in comparing multiple samples in a single LC/ESI-MS analysis. The potential of this strategy for defining glycomic differences in blood serum samples representing different esophageal diseases was explored.LC/ESI-MS comparative glycomic profiling of isotopically permethylated N-glycans derived from biological samples and glycoproteins reliably defined glycan changes associated with biological conditions or glycoproteins expression. As a biological application, this strategy permitted the reliable quantification of glycomic changes associated with different esophageal diseases, including high grade dysplasia, Barrett's disease, and esophageal adenocarcinoma.
Project description:Fatty acids (FAs) are essential components in cells and are involved in many cellular activities. Abnormal FA metabolism has been reported to be related to human diseases such as cancer and cardiovascular diseases. Identification and quantification of FAs provide insights into their functions in biological systems, but it is very challenging to analyze them due to their structures and properties. In this work, we developed a novel method by integrating FAs tagged with stable isotope labeled aminoxy tandem mass tags (aminoxyTMTs) and mass spectrometric analysis in the positive mode. On the basis of their structures, the aminoxyTMT reagents reacted with the carboxylic acid group of the FAs, resulting in an amine group with high proton affinity covalently attached to the analytes. This enabled the analysis of FAs under the positive electrospray ionization-mass spectrometry (ESI-MS) mode, which is normally more popular and sensitive compared to the negative mode. More importantly, the multiplexed TMT tags allowed us to quantify FAs from several samples simultaneously, which increased the experimental throughput and quantification accuracy. FAs extracted from three types of breast cells, i.e., MCF 10A (normal), MCF7 (minimally invasive) and MDA-MB-231 (highly invasive) cells, were labeled with the six-plexed aminoxyTMTs and quantified by LC-MS/MS. The results demonstrated that the abundances of some FAs, such as C22:5 and C20:3, were markedly increased in MCF7 and MDA-MB-231 cancer cells compared to normal MCF 10A cells. For the first time, aminoxyTMT reagents were exploited to label FAs for their identification and quantification in complex biological samples in the positive MS mode. The current method enabled us to confidently identify FAs and to accurately quantify them from several samples simultaneously. Because this method does not have sample restrictions, it can be extensively applied for biological and biomedical research.
Project description:Protein glycosylation mediates a wide range of cellular processes, affecting development and disease in mammals. Deciphering the "glycocodes" requires rapid, sensitive and in-depth characterization of diverse glycan structures derived from biological samples. In this study, we described a two-step derivatization strategy termed linkage-specific sialic acid permethylation (SSAP) consisting of dimethylamination and permethylation for the improved profiling of glycosylation by matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS). High linkage-specificity (?99%) of SSAP to both the two most common forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), permitted direct discrimination of ?2,3- and ?2,6-linked sialic acids in MALDI-TOF MS. The enhanced intensity (>10-fold) and increased detection limit (>10-fold) of derivatized glycans were valued for sensitive glycomics. Moreover, the good compatibility and reaction efficiency of the two steps of SSAP allowed rapid sample preparation (<2 h), benefiting robust analysis of glycans in a high-throughput manner. The SSAP strategy was further applied to investigate the protein glycosylation of human serum associated with rheumatoid arthritis (RA). It was demonstrated that the relative abundances of individual glycans were different in RA negative and RA positive samples, and meanwhile the RA patient/control ratios of both ?2,3- and ?2,6-sialylated glycans tended to elevate accompanied with the increase of sialylation. Those findings of the glycosylation changes occurred in human serum protein may contribute to the diagnosis of RA. Herein, SSAP derivatization combined with MALDI-TOF MS exhibits unique advantages for glycomic analysis and shows potential in glycosylation profiling of therapeutic proteins and clinical glycan biomarker discovery.
Project description:Colorectal cancer is the fourth most prevalent cancer in the United States, yet there are no reliable noninvasive early screening methods available. Serum-based glycomic profiling has the necessary sensitivity and specificity to distinguish disease states and provide diagnostic potential for this deadly form of cancer. We applied microchip electrophoresis and MALDI-TOF-MS-based glycomic procedures to 20 control serum samples and 42 samples provided by patients diagnosed with colorectal cancer. Within the identified glycans, the position of fucose units was located to quantitate possible changes of fucosyl isomeric species associated with the pathological condition. MALDI-MS data revealed several fucosylated tri- and tetra-antennary glycans which were significantly elevated in their abundance levels in the cancer samples and distinguished the control samples from the colorectal cancer cohort in the comprehensive profiles. When compared to other cancers studied previously, some unique changes appear to be associated with colorectal cancer, being primarily associated with fucosyl isomers. Through MS and microchip electrophoresis-based glycomic methods, several potential biomarkers were identified to aid in the diagnosis and differentiation of colorectal cancer. With its unique capability to resolve isomers, microchip electrophoresis can yield complementary analytical information to MS-based profiling.
Project description:As a common post-translational modification, protein glycosylation plays an important role in many biological processes, and it is known to be associated with human diseases. Mass spectrometry (MS)-based glycomic profiling techniques have been developed to measure the abundances of glycans in complex biological samples and applied to the discovery of putative glycan biomarkers. To automate the annotation of glycomic profiles in the liquid chromatography-MS (LC-MS) data, we present here a user-friendly software tool, MultiGlycan, implemented in C# on Windows systems. We tested MultiGlycan by using several glycomic profiling datasets acquired using LC-MS under different preparations and show that MultiGlycan executes fast and generates robust and reliable results.MultiGlycan can be freely downloaded at http://darwin.informatics.indiana.edu/MultiGlycan/.Supplementary data are available at Bioinformatics online.