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.
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 esophagus diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.
The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of ϩ ions. PMP-and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M ϩ Na] ϩ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Glycosylation is also highly sensitive to alterations of cellular function, and altered protein glycosylation is diagnostic of a number of disease states including, for example, rheumatoid arthritis and cancer [3]. Therefore, the growing interest in understanding the biological functions of carbohydrates has stimulated the development of methods for their improved analysis.In order to detect saccharides by UV/fluorescence and also to ease their characterization by mass spectrometry (MS), introducing a chromophore into the molecules has received increasing attention. In this respect, a large number of derivatization procedures for mono-and oligosaccharides have been described in the literature, most of which have been detailed in a recent review [4]. For example, 2-aminoacridine (2-AMAC) as a sensitive fluorophore, has been used for the detection of monosaccharides, and of neutral and charged oligosaccharides by electrospray ionization (ESI) and matrix-
N-Linked glycans derived from human and bovine alpha1-acid glycoprotein, as well as chicken egg white albumin, were analyzed by MALDI-TOF mass spectrometry using a novel MALDI matrix consisting of 2,5-dihydroxybenzoic acid (DHB) and aniline. A significant increase in signal was observed for these oligosaccharides relative to the signal obtained when unmodified DHB was used as a matrix for the same set of samples. The use of aniline/DHB matrix also led to facile on-target derivatization of the glycans via nonreductive amination, as aniline was found to form a stable Schiff base with the reducing end GlcNAc residue without the need for prolonged incubation periods and elevated temperatures. Both native and derivatized glycans ionized as sodium adducts and had similar MS/MS fragmentation patterns consisting mainly of Y/B-cleavage ions. In our experiments, we obtained evidence for persistence of the derivatization reaction in the solid phase; i.e., the reaction appeared to be taking place even after the sample-matrix spot had dried. This is the first report on such solid-phase on-target derivatization of carbohydrates for subsequent analysis by MALDI mass spectrometry.
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