Shin-ichirou Kawabata

In-Depth Structural Characterization of N-Linked Glycopeptides Using Complete Derivatization for Carboxyl Groups Followed by Positive- and Negative-Ion Tandem Mass Spectrometry

Tandem mass spectrometry (MS/MS or MS(n)) is a powerful tool for characterizing N-linked glycopeptide structures. However, it is still difficult to obtain detailed structural information on the glycan moiety directly from glycopeptide ions. Here, we propose a new method for in-depth analysis of the glycopeptide structure using MS/MS. This method involves complete derivatization of carboxyl groups in glycopeptides. Methylamidation using PyAOP as a condensing reagent has been optimized for derivatizing all carboxyl groups in glycopeptides. By derivatizing carboxyl groups on the peptide moiety (i.e., Asp, Glu, and C-terminus), the glycopeptides efficiently produce informative glycan fragment ions, including the nonreducing end of the glycan moiety under negative-ion collision-induced dissociation (CID) conditions. These glycan fragment ions can define detailed structural features on the glycan moiety (e.g., the specific composition of the two antennae, the location of fucose residues, and the presence/absence of bisecting GlcNAc residues). For sialylated glycopeptides, carboxyl groups on sialic acid residues are simultaneously derivatized using methylamidation, suppressing preferential loss of residues during MS analysis. As a result, both sialylated and nonsialylated glycopeptides can be analyzed in the same manner. Positive-ion CID of methylamine-derivatized glycopeptides mainly provides information on peptide sequence and glycan composition, whereas negative-ion CID provides in-depth structural information on the glycan moiety. The derivatization step can be readily incorporated into conventional pretreatment for glycopeptide MS analysis without loss of sensitivity, making derivatization suitable for practical use.

Sensitive analyses of neutral N-glycans using anion-doped liquid matrix G3CA by negative-ion matrix-assisted laser desorption/ionization mass spectrometry.

Negative-ion fragmentation of N-glycans has been proven to be more informative than that of positive-ion. In particular, it defines structural features such as the specific composition of the two antennae and the location of fucose. However, negative-ion formation of neutral N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) remains a challenging task, and the detection limit of N-glycans in negative-ion mode is merely at the subpicomole level. Thus, practical applications are limited. In this study, combinations of five liquid matrices and nine anions were used to ionize N-glycans as anionic adducts, and their performances for sensitive analyses were evaluated. The best results were obtained with anion-doped liquid matrix G(3)CA, which consists of p-coumaric acid and 1,1,3,3-tetramethylguanidine; the detection limits of anion adducted N-glycans were 1 fmol/well for NO(3)(-), and 100 amol/well for BF(4)(-). Negative-ion MS(2) spectra of 1 fmol N-glycans were successfully acquired with a sufficient signal-to-noise ratio and were quite useful for MS-based structural determination. The anion-doped G(3)CA matrix opens the way for sensitive and rapid analysis of neutral N-glycans in negative-ion MALDI at a low femtomole level.

3-Aminoquinoline/p-coumaric acid as a MALDI matrix for glycopeptides, carbohydrates, and phosphopeptides.

Glycosylation and phosphorylation are important post-translational modifications in biological processes and biomarker research. The difficulty in analyzing these modifications is mainly their low abundance and dissociation of labile regions such as sialic acids or phosphate groups. One solution in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is to improve matrices for glycopeptides, carbohydrates, and phosphopeptides by increasing the sensitivity and suppressing dissociation of the labile regions. Recently, a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) (3-AQ/CHCA), introduced by Kolli et al. in 1996, has been reported to increase sensitivity for carbohydrates or phosphopeptides, but it has not been systematically evaluated for glycopeptides. In addition, 3-AQ/CHCA enhances the dissociation of labile regions. In contrast, a liquid matrix 1,1,3,3-tetramethylguanidium (TMG, G) salt of p-coumaric acid (CA) (G3CA) was reported to suppress dissociation of sulfate groups or sialic acids of carbohydrates. Here we introduce a liquid matrix 3-AQ/CA for glycopeptides, carbohydrates, and phosphopeptides. All of the analytes were detected as [M + H](+) or [M - H](-) with higher or comparable sensitivity using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-dihydroxybenzoic acid (2,5-DHB). The sensitivity was increased 1- to 1000-fold using 3-AQ/CA. The dissociation of labile regions such as sialic acids or phosphate groups and the fragmentation of neutral carbohydrates were suppressed more using 3-AQ/CA than using 3-AQ/CHCA or 2,5-DHB. 3-AQ/CA was thus determined to be an effective MALDI matrix for high sensitivity and the suppression of dissociation of labile regions in glycosylation and phosphorylation analyses.

Structural analysis of N-glycans by the glycan-labeling method using 3-aminoquinoline-based liquid matrix in negative-ion MALDI-MS.

Negative-ion fragmentation of underivatized N-glycans has been proven to be more informative than positive-ion fragmentation. Fluorescent labeling via reductive amination is often employed for glycan analysis, but little is known about the influence of the labeling group on negative-ion fragmentation. We previously demonstrated that the on-target glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) liquid matrix enables highly sensitive, rapid, and quantitative N-glycan profiling analysis. The current study investigates the suitability of 3AQ-labeled N-glycans for structural analysis based on negative-ion collision-induced dissociation (CID) spectra. 3AQ-labeled N-glycans exhibited simple and informative CID spectra similar to those of underivatized N-glycans, with product ions due to cross-ring cleavages of the chitobiose core and ions specific to two antennae (D and E ions). The interpretation of diagnostic fragment ions suggested for underivatized N-glycans could be directly applied to the 3AQ-labeled N-glycans. However, fluorescently labeled N-glycans by conventional reductive amination, such as 2-aminobenzamide (2AB)- and 2-pyrydilamine (2PA)-labeled N-glycans, exhibited complicated CID spectra consisting of numerous signals formed by dehydration and multiple cleavages. The complicated spectra of 2AB- and 2PA-labeled N-glycans was found to be due to their open reducing-terminal N-acetylglucosamine (GlcNAc) ring, rather than structural differences in the labeling group in the N-glycan derivative. Finally, as an example, the on-target 3AQ labeling method followed by negative-ion CID was applied to structurally analyze neutral N-glycans released from human epidermal growth factor receptor type 2 (HER2) protein. The glycan-labeling method using 3AQ-based liquid matrix should facilitate highly sensitive quantitative and qualitative analyses of glycans.

Alkylated Dihydroxybenzoic Acid as a MALDI Matrix Additive for Hydrophobic Peptide Analysis

Hydrophobic peptides are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) because the majority of MALDI matrixes are hydrophilic and therefore have a low affinity for hydrophobic peptides. Here, we report on a novel matrix additive, o-alkylated dihydroxybenzoic acid (ADHB), which is a 2,5-dihydroxybenzoic acid (DHB) derivative incorporating a hydrophobic alkyl chain on a hydroxyl group to improve its affinity for hydrophobic peptides, thereby improving MALDI-MS sensitivity. The addition of ADHB to the conventional matrix α-cyano-4-hydroxycinnamic acid (CHCA) improved the sensitivity of hydrophobic peptides 10- to 100-fold. The sequence coverage of phosphorylase b digest was increased using ADHB. MS imaging indicated that hydrophobic peptides were enriched in the rim of a matrix/analyte dried spot when ADHB was used. In conclusion, the addition of ADHB to the standard matrix led to improved sensitivity of hydrophobic peptides by MALDI-MS.

Post‐source Decay Fragment Spectra of Cyclomalto‐octaose and Branched Cyclomalto‐hexaose by Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry

gamma-Cyclodextrin, maltosyl-alpha-cyclodextrin and diglucosyl-alpha-cyclodextrin were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. All of these compounds have the same molecular weight (M.W. = 1297.15) and consist of only D-glucopyranose. From a comparison of the intensities in the post-source decay (PSD) fragment spectra of these cyclodextrin derivatives, correlation between the chemical structures and the relative intensities in the PSD fragment ions was found. The correlation is considered to be caused by the difference in the number of cleavage sites at the glycosyl binding. It was found that the intensity of the PSD ion resulting from one cleavage is higher than that resulting from two cleavages at a glycosyl bond. The results show that PSD fragment-ion spectrum method used in MALDI-TOF mass spectrometry is a very powerful technique for the structural analyses of the sugar-substituted cyclodextrins.

Analysis of Glycosidic Linkages in Saccharide Compounds by Post-source Decay Fragment Methods in Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy

Maltotriosyl- and panosyl-alpha-cyclodextrins and the nonaose of pullulan were analyzed by post-source decay (PSD) fragment methods of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. By the mass number analysis, it was found that all of the PSD fragment ions were produced by cleavages of glycosidic linkages. Comparison of the relative intensities of the ions in those compounds enabled us to distinguish two kinds of glycosidic linkages, alpha 1-4 and alpha 1-6, by MALDI-TOFMS with a new type of ion reflector: the curved field reflectron.

Fragmentation Characteristics of Deprotonated N-linked Glycopeptides: Influences of Amino Acid Composition and Sequence

AbstractGlycopeptide structural analysis using tandem mass spectrometry is becoming a common approach for elucidating site-specific N-glycosylation. The analysis is generally performed in positive-ion mode. Therefore, fragmentation of protonated glycopeptides has been extensively investigated; however, few studies are available on deprotonated glycopeptides, despite the usefulness of negative-ion mode analysis in detecting glycopeptide signals. Here, large sets of glycopeptides derived from well-characterized glycoproteins were investigated to understand the fragmentation behavior of deprotonated N-linked glycopeptides under low-energy collision-induced dissociation (CID) conditions. The fragment ion species were found to be significantly variable depending on their amino acid sequence and could be classified into three types: (i) glycan fragment ions, (ii) glycan-lost fragment ions and their secondary cleavage products, and (iii) fragment ions with intact glycan moiety. The CID spectra of glycopeptides having a short peptide sequence were dominated by type (i) glycan fragments (e.g., 2,4AR, 2,4AR-1, D, and E ions). These fragments define detailed structural features of the glycan moiety such as branching. For glycopeptides with medium or long peptide sequences, the major fragments were type (ii) ions (e.g., [peptide + 0,2X0–H]– and [peptide–NH3–H]–). The appearance of type (iii) ions strongly depended on the peptide sequence, and especially on the presence of Asp, Asn, and Glu. When a glycosylated Asn is located on the C-terminus, an interesting fragment having an Asn residue with intact glycan moiety, [glycan + Asn–36]–, was abundantly formed. Observed fragments are reasonably explained by a combination of existing fragmentation rules suggested for N-glycans and peptides. Figureᅟ

An optimized matrix-assisted laser desorption/ionization sample preparation using a liquid matrix, 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid, for phosphopeptides

RATIONALE A liquid matrix, 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA), introduced by Kolli et al. in 1996 for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported for peptides and proteins, oligonucleotides, oligosaccharides, and glycopeptides. However, it has not been validated for phosphopeptides. METHODS We optimized sample preparation using 3-AQ/CHCA for phosphopeptides. The sensitivity of six phosphopeptide species as isolated or in digests was systematically evaluated by using MALDI-quadropole ion trap (QIT)-time of flight (TOF) MS in positive and negative ion modes, and compared with the conventional methods using a solid matrix, 2,5-dihydroxybenzoic acid (2,5-DHB). RESULTS The sensitivity of mono- and tetraphosphopeptides was improved 10- to 10 000-fold with the optimized preparation method using 3-AQ/CHCA compared with the conventional methods using 2,5-DHB. Improvement by 3-AQ/CHCA itself was 10-fold. Adding ammonium dihydrogen phosphate or an analyte solvent composition was also effectively improved the sensitivity. Phosphopeptides in isolated form or in digests were detected at femto- or subfemtomole levels. CONCLUSIONS Sensitivity of phosphopeptides was improved by the optimized sample preparation method using 3-AQ/CHCA compared with the conventional method using 2,5-DHB. The validation of 3-AQ/CHCA for phosphopeptides was systematically confirmed, expanding the potential of this matrix to phosphoproteomics.

Laser Desorption/Ionization Mass Spectrometry (LDI-MS) of Lipids with Iron Oxide Nanoparticle-Coated Targets.

Iron oxide nanoparticle (NP)-coated target plates were employed for the direct detection and analysis of low molecular weight lipids by laser desorption/ionization (LDI) mass spectrometry (MS). We have demonstrated that the use of the iron oxide NP-coated target provides a simple, direct, and rapid detection method for lipid standards and epidermal surface lipids without any cumbersome sample pretreatment as well as mass spectra that are free of background matrix peaks. Lipid standards (1-stearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol, 1,2-distearoyl-sn-glycero-3-phosphocholine) were detected as either protonated or cationated species. Clean MS/MS spectra for each lipid were also successfully obtained. Pre-MS surface cleaning of the target plates with UV-ozone treatment successfully removed organic contaminants that would interfere with the mass spectra especially in the low molecular weight region. Preliminary application of the presented target plate to the detection of endogenous lipids in latent fingerprints showed promising results and for potential use in the visualization and chemical composition determination of latent fingerprints by nanoparticle assistance.