Quantitative profiling of O-glycans by electrospray ionization- and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry after in-gel derivatization with isotope-coded 1-phenyl-3-methyl-5-pyrazolone.

The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d0/d5-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization.

Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

Rationale: Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively.

Quantitative fingerprinting of O-linked glycans released from proteins using isotopic coded labeling with deuterated 1-phenyl-3-methyl-5-pyrazolone.

Investigation of oligosaccharides attached to proteins as post-translational modification remains an important research field in the area of glycoproteomics as well as in biotechnology. The development of new tools for qualitative and quantitative analysis of glycans has gained high importance in recent years. This is particularly true with O-glycans for which quantitative data are still underrepresented in literature.

Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase.

Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins.

Separation and identification of glycoforms by capillary electrophoresis with electrospray ionization mass spectrometric detection.

Capillary electrophoresis (CE) is a resourceful and versatile separation method for the analysis of complex carbohydrate mixtures. In combination with electrospray ionization (ESI) mass spectrometry (MS), CE enables fast, sensitive, and efficient separations for the accurate identification of a large variety of glycoform mixture types. In this chapter several reliable off- and on-line CE-based methods for the analysis of glycoforms with ESI MS/MS are presented.

Use of a novel ionic liquid matrix for MALDI-MS analysis of glycopeptides and glycans out of total tryptic digests.

In this study, we investigated a novel ionic liquid matrix (ILM), namely, the 1,1,3,3-tetramethylguanidinium salt of 2,4,6-trihydroxyacetophenone (THAP). This matrix[1,1,3,3-tetramethylguanidinium 2,4,6-trihydroxyacetophenone (GTHAP)] turned out to be well suited for the matrix-assisted laser desorption/ionization mass spectrometric analysis of glycopeptides and glycans, and overcame the well-known ionization suppression of carbohydrate structures in the presence of peptides. The matrix was evaluated by two different series of experiments, in each case in comparison with the crystalline THAP matrix.

Capillary isoelectric focusing hyphenated to single- and multistage matrix-assisted laser desorption/ionization-mass spectrometry using automated sheath-flow-assisted sample deposition.

In this paper CIEF combined with MALDI-MS is described using a sheath-liquid-assisted automatic sample deposition from the separation capillary onto a MALDI target. Sample/matrix preparation techniques on the target resembling the dried droplet and the thin layer methods were evaluated in the context of the automatic spotting. Volatile buffers were used as IEF catholyte solutions.