Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer.

Targeted mass spectrometric analysis is widely employed across various omics fields. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and small molecules. Selected reaction monitoring and multiple reaction monitoring (MRM) have been a method of choice for targeted structural studies of biomolecules. However, innovations in instrument designs have led to the development of parallel reaction monitoring (PRM). PRM detects all product ions simultaneously rather than optimizing/preselecting the target glycan transitions, simplifying the analytical workflow. By reducing background interference, increasing selectivity/specificity, and improving data quality, PRM allows reliable quantification of target glycans in complex matrices. PRM can also improve sensitivity for detecting low-abundance target glycans and reduce low-level limit of quantification values with an improved S/N ratio. PRM's advantages are attributed to the development of sensitive and highly selective mass analyzers, orbitrap, and time of flight. In this study, we developed a sensitive PRM method for the quantitative analysis of permethylated N-glycans, an important class of disease biomarkers, using a quadrupole-orbitrap hybrid mass spectrometer. Pooled human cerebrospinal fluid was used for the study as a source of permethylated N-glycans. The method illustrates the fragmentation of N-glycans at different collision energies as well as the optimization of collision energy. The method also detects low-abundance N-glycans more efficiently than MRM. This study is the first attempt to develop a sensitive PRM-based method to analyze permethylated N-glycans.

Determination of Isomeric Glycan Structures by Permethylation and Liquid Chromatography-Mass Spectrometry (LC-MS).

The existence of glycans in isomeric forms is responsible for the multifariousness of their properties and biological functions. Their altered expression has been associated with various diseases and cancers. Analysis of native glycans is not very sensitive due to the low ionization efficiency of glycans. These facts necessitate their comprehensive structural studies and establishes a high demand for sensitive and reliable techniques. In this chapter, we discuss the strategies for effective separation and identification of permethylated isomeric glycans. The sample preparation for permethylated glycans derived from model glycoproteins and complex biological samples, analyzed using LC-MS/MS, is delineated. We introduce protein extraction and release of glycans, followed by strategies to purify the released glycans, which are reduced and permethylated to improve ionization efficiency and stabilize sialic acid residues. High-temperature LC-based separation on PGC (porous graphitized carbon) column is conducive to isomeric separation of glycans and allows their sensitive identification and quantification using MS/MS.

Mesoporous Graphitized Carbon Column for Efficient Isomeric Separation of Permethylated Glycans.

Post-translational modifications are vital aspects of functional proteins. Therefore, it is critical to understand their roles in biological processes. Glycosylation is particularly challenging to study among these modifications due to the heterogeneity displayed by the glycans in terms of their isomers. Thus, researchers continue to strive for the development of efficient liquid chromatography techniques for isomeric separation of glycans. Porous graphitized carbon (PGC) nano column has been one of the most widely used columns for this purpose, but poor stability and lack of reproducibility led to its discontinuation. In our endeavor to find an alternative stationary phase for isomeric glycan separation, we tested the mesoporous graphitized carbon (MGC) material. Unprecedentedly, satisfactory results were obtained with a column only 1 cm long, which was tested on permethylated N-glycans derived from model glycoproteins as well as biological samples. The column was found to be reproducible across months as well as across different column preparations. Additionally, to decrease the dead volume and attain a better resolution, MGC was utilized to pack a 1 cm length of a pulled capillary nanospray emitter and again demonstrated efficient isomeric separation. Thus, MGC proved to be a suitable stationary phase to obtain efficient isomeric separation of permethylated N-glycans with 1 cm-long packing length, in both capillary columns and packed nanospray emitters.

Glucose unit index (GUI) of permethylated glycans for effective identification of glycans and glycan isomers.

Retention time is the most common and widely used criterion to report the separation of glycans using Liquid Chromatography (LC), but it varies widely across different columns, instruments and laboratories. This variation is problematic when inter-laboratory data is compared. Furthermore, it influences reproducibility and hampers efficient data interpretation. In our endeavor to overcome this variance, we propose the use of the Glucose Unit Index (GUI) on C18 and PGC column-based separation of reduced and permethylated glycans. GUI has previously been utilized for retention time normalization of native and labeled glycans. We evaluated this method with reduced and permethylated glycans derived from model glycoproteins fetuin and ribonuclease B (RNase B), and then implemented it to human blood serum to generate C18 and PGC column-based isomeric glycan libraries. GUI values for glycan compositions were calculated with respect to the glucose units derived from dextrin, which was employed as an elution standard. The GUI values were validated on three different LC systems (UltiMate 3000 Nano UHPLC systems) in two laboratories to ensure the reliability and reproducibility of the method. Applicability on real samples was demonstrated using human breast cancer cell lines. A total of 116 permethylated N-glycans separated on a C18 column and 134 glycans separated on a PGC column were compiled in a library. Overall, the established GUI method and the demonstration of reproducible inter- and intra-laboratory GUI values would aid the future development of automated glycan and isomeric glycan identification methods.

Characterization of glycan isomers using magnetic carbon nanoparticles as a MALDI co-matrix.

Matrix-assisted laser desorption ionization-in source decay (MALDI-ISD) analysis is a useful technique in the structural analysis of glycans. Our recent publication demonstrated that magnetic carbon nanoparticles (MCNPs), used as a MALDI co-matrix, significantly enhanced ISD efficiency for glycomic analysis by MALDI-TOF. In this study, MCNPs were used for the structural study of isomeric glycans. Results from the standard glycans confirmed easy distinction of positional and linkage isomers without the need for further derivatization of glycan molecules. Extensive glycosidic and cross-ring fragmented ions provided different fragment patterns for various glycan isomers. Core- and branch-fucosylated isomers were distinguished by several unique ions, and pseudo-MS3 data were used to recognize the fucosylated branch. Although no diagnostic fragment ion was observed for 2,3- and 2,6-linked sialic acid isomers, their MALDI-ISD patterns were found to be significantly different (P < 0.05). Furthermore, the method introduced in this study could not only be used for the identification of glycan isomers but has also proved effective for the isomeric structural confirmation of gangliosides. GD1a and GD1b gangliosides were easily distinguished by the diagnostic ion originated from GD1a, produced by Z4αZ2ß cleavages. Moreover, liquid chromatography coupled with MALDI-TOF was applied to analyze N-glycan isomers derived from a pooled human blood serum sample, providing an alternative method of isomeric glycomic analysis of biological specimens.

Clinical application of quantitative glycomics.

INTRODUCTION: Aberrant glycosylation has been associated with many diseases. Decades of research activities have reported many reliable glycan biomarkers of different diseases which enable effective disease diagnostics and prognostics. However, none of the glycan markers have been approved for clinical diagnosis. Thus, a review of these studies is needed to guide the successful clinical translation. Area covered: In this review, we describe and discuss advances in analytical methods enabling clinical glycan biomarker discovery, focusing only on studies of released glycans. This review also summarizes the different glycobiomarkers identified for cancers, Alzheimer's disease, diabetes, hepatitis B and C, and other diseases. Expert commentary: Along with the development of techniques in quantitative glycomics, more glycans or glycan patterns have been reported as better potential biomarkers of different diseases and proved to have greater diagnostic/diagnostic sensitivity and specificity than existing markers. However, to successfully apply glycan markers in clinical diagnosis, more studies and verifications on large biological cohorts need to be performed. In addition, faster and more efficient glycomic strategies need to be developed to shorten the turnaround time. Thus, glycan biomarkers have an immense chance to be used in clinical prognosis and diagnosis of many diseases in the near future.

Advances in mass spectrometry-based glycoproteomics.

Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host-pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and poor ionization efficiency of glycopeptides. Therefore, the development of sensitive and accurate approaches to characterize protein glycosylation is crucial. The identification and characterization of protein glycosylation by MS is referred to as the field of glycoproteomics. Methods such as enrichment, metabolic labeling, and derivatization of glycopeptides in conjunction with different MS techniques and bioinformatics tools, have been developed to achieve an unequivocal quantitative and qualitative characterization of glycoproteins. This review summarizes the recent developments in the field of glycoproteomics over the past 6 years (2012 to 2018).

Advances in mass spectrometry-based glycomics.

The diversification of the chemical properties and biological functions of proteins is attained through posttranslational modifications, such as glycosylation. Glycans, which are covalently attached to proteins, play a vital role in cell activities. The microheterogeneity and complexity of glycan structures associated with proteins make comprehensive glycomic analysis challenging. However, recent advancements in mass spectrometry (MS), separation techniques, and sample preparation methods have primarily facilitated structural elucidation and quantitation of glycans. This review focuses on describing recent advances in MS-based techniques used for glycomic analysis (2012-2018), including ionization, tandem MS, and separation techniques coupled with MS. Progress in glycomics workflow involving glycan release, purification, derivatization, and separation will also be highlighted here. Additionally, the recent development of quantitative glycomics through comparative and multiplex approaches will also be described.

Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics.

More than 50% of all known proteins are glycosylated, which is critical for many biological processes such as protein folding and signal transduction. Glycosylation has proven to be associated with different mammalian diseases such as breast and liver cancers. Therefore, characterization of glycans is highly important to facilitate a better understanding of the development and progression of many human diseases. Although matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) offers several advantages such as ease of operation and short analysis times, however, due to the complexity of glycan structures and their low ionization efficiency, there are still challenges that need to be addressed to achieve sensitive glycan analysis. Here, magnetic carbon nanocomposites (CNPs@Fe3O4 NCs) were used as a new MALDI matrix or co-matrix for the analysis of glycans derived from different model glycoproteins and human blood serum samples. The addition of CNPs@Fe3O4 NCs to the matrix significantly enhanced glycan signal intensity by several orders of magnitude, and effectively controlled/reduced/eliminated in-source decay (ISD) fragmentation. The latter was attained by modulating CNPs@Fe3O4 NCs concentrations and allowed the simultaneous study of intact and fragmented glycans, and pseudo-MS3 analysis. Moreover, CNPs@Fe3O4 NCs was also effectively employed to desalt samples directly on MALDI plate, thus enabling direct MALDI-MS analysis of unpurified permethylated glycans derived from both model glycoproteins and biological samples. On-plate desalting enhanced sensitivity by reducing sample loss. Graphical abstract ᅟ.

A carbon nanoparticles-based solid-phase purification method facilitating sensitive MALDI-MS analysis of permethylated N-glycans.

In recent decades, MALDI-MS has been extensively used for the analysis of glycans. However, native glycans usually have low ionization efficiency in MS, which hinders the direct analysis. Permethylation of glycans is a solution for this issue, but a significant amount of salt is introduced during this process, which can further suppress the MS signals. Thus, it is necessary to purify the glycans prior to MALDI-MS analysis. In this study, we developed a carbon nanoparticles-based solid-phase purification method to enable direct MALDI-MS analysis of permethylated glycans. Two carbon nanomaterials, carbon nanoparticles (CNPs) and graphene nanosheets (GNs), and two conventional carbon materials, activated charcoal and porous graphitic carbon (PGC), were investigated as sorbents to purify permethylated N-glycans derived from ribonuclease B and fetuin. The results confirmed the superior performance of CNPs over the other carbon materials. Additionally, our method was also employed to purify glycans released from human sera in different esophageal disease stages. The obtained data confirmed 16 and 18 structures in adenocarcinoma and Barret's sera with significantly different relative intensities versus disease-free sera. Comparing the performance of CNPs-based solid-phase purification method employed in this study to online purification suggested more than 97% recovery rate. The results of this study demonstrate that CNPs have the potential to be a better alternative to existing solid-phase purification sorbents.

Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples.

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS2 and pseudo-MS3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

Recent advances in mass spectrometric analysis of glycoproteins.

Glycosylation is one of the most common posttranslational modifications of proteins that plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry (MS) in conjunction with different separation methods, such as capillary electrophoresis (CE), ion mobility (IM), and high performance liquid chromatography (HPLC) has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

Synthesis of palladium nanoparticles on organically modified silica: application to design of a solid-state electrochemiluminescence sensor for highly sensitive determination of imipramine.

Organically modified silica substrate containing amine and vinyl functional groups were used for reduction and stabilization of palladium nanoparticles. Uniform spherical nanoparticles of palladium with average diameter of 10 nm were formed on silica substrate by direct contact of the substrate with an aqueous solution of palladium precursor, without the addition of any chemical reducer. Moreover, a sensitive and selective solid state electrochemiluminescence sensor was fabricated for the determination of imipramine, based on Ru(bpy)3(2+)-palladium nanoparticles doped carbon ionic liquid electrode. In this process, imipramine acts as a co-reactant for Ru(bpy)3(2+). It is believed that the enhancement of the electrochemiluminescence signal in the presence of palladium nanoparticles in the composite is due to palladium catalytic effect on electrochemical and also chemical process involved in formation of Ru(byp)3(2+)*. In addition, the results confirmed that, the rigid composite electrode shows the characteristic of microelectrode arrays. The proposed method was applied to the determination of imipramine in tablets and urine samples. The electrochemiluminescence intensity showed good linearity with the imipramine concentration from 1-100 pM, with a detection limit of 0.1 pM.