Takashi Nishikaze

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.

Derivatization with 1-pyrenyldiazomethane enhances ionization of glycopeptides but not peptides in matrix-assisted laser desorption/ionization mass spectrometry.

Glycoproteomics holds the promise of new advances in medical technology. However, mass spectrometry has limitations for the structural determination of glycosylated peptides because the hydrophilic nature of the oligosaccharide moiety in glycopeptides is disadvantageous for ionization, and glycopeptides ionize much less readily than nonglycosylated peptides. Therefore, conventional proteomics tools cannot detect altered glycosylation on proteins. Here, we describe an on-plate pyrene derivatization method using 1-pyrenyldiazomethane for highly sensitive matrix-assisted laser/desorption ionization-tandem mass spectrometry (MALDI-MS(n)) of glycopeptides in amounts of less than 100 fmol. This derivatization is unique, as the pyrene groups are easily released from glycopeptides during ionization when 2,5-dihydroxybenzoic acid is used as a matrix. As a result, most ions are observed as the underivatized form on the spectra. At the same time, pyrene derivatization dramatically reduces the ionization of peptides. Thus, for glycopeptides in a mixture of abundant peptides, we could obtain MS spectra in which the signals of glycopeptides were intense enough for subjection to MS(n) in order to determine the structures of both glycan and peptide. Finally, we show that the glycopeptides derived from as little as 1 ng of prostate specific antigen can be detected by this method.

Influence of Charge State and Amino Acid Composition on Hydrogen Transfer in Electron Capture Dissociation of Peptides

Although conventional N-Cα bond cleavage in electron capture dissociation (ECD) of multiply-charged peptides generates a complementary c′ and z′ fragment pair, the N-Cα cleavage followed by hydrogen transfer from c′ to z′ fragments produces other fragments, namely c′ and z′. In this study, the influence of charge state and amino acid composition on hydrogen transfer in ECD is described using sets of peptides. Hydrogen transferred ionic species such as c′ and z′ were observed in ECD spectra of doubly-protonated peptides, while the triply-protonated form did not demonstrate hydrogen transfer. The extent of hydrogen transfer in ECD of doubly-protonated peptides was dependent on constituent amino acids. The ECD of doubly-protonated peptides possessing numerous basic sites showed extensive hydrogen transfer compared with ECD of less basic peptides. The extent of hydrogen transfer is discussed from the viewpoints of the structure of peptide ions, the possibility of internal hydrogen bonding and intermediate lifetime of complex [c′+z′].

Negative-ion MALDI-MS2 for discrimination of α2,3- and α2,6-sialylation on glycopeptides labeled with a pyrene derivative ☆

Here, we propose a novel method for the discrimination of α2,3- and α2,6-sialylation on glycopeptides. To stabilize the sialic acids, the carboxyl moiety on the sialic acid as well as the C-terminus and side chain of the peptide backbone were derivatized using 1-pyrenyldiazomethane (PDAM). The derivatization can be performed on the target plate for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), thereby avoiding complicated and time-consuming purification steps. After the on-plate PDAM derivatization, samples were subjected to negative-ion MALDI-MS using 3AQ-CHCA as a matrix. Deprotonated ions of the PDAM-derivatized form were detected as the predominant species without loss of sialic acid. The negative-ion collision-induced dissociation (CID) of PDAM-derivatized isomeric sialylglycopeptides, derived from hen egg yolk, showed characteristic spectral patterns. These data made it possible to discriminate α2,3- and α2,6-sialylation. In addition, sialyl isomers of a glycan with an asparagine could be discriminated based on their CID spectra. In brief, the negative-ion CID of PDAM-derivatized glycopeptides with α2,6-sialylation gave an abundant (0,2)A-type product ion, while that with α2,3-sialylation furnished a series of (2,4)A/Y-type product ions with loss of sialic acids. The unique fragmentation behavior appears to be derived from the difference of pyrene binding positions after ionization, depending on the type of sialylation. Thus, we show that on-plate PDAM derivatization followed by negative-ion MALDI-MS(2) is a simple and robust method for the discrimination of α2,3- and α2,6-sialylation on glycopeptides.

Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.

Hydrogen Attachment/Abstraction Dissociation (HAD) of Gas-Phase Peptide Ions for Tandem Mass Spectrometry

Dissociation of gas-phase peptide ions through interaction with low-energy hydrogen (H) radical (∼0.15 eV) was observed with a quadrupole ion trap mass spectrometry. The H radical generated by thermal dissociation of H2 molecules passing through a heated tungsten capillary (∼2000 °C) was injected into the ion trap containing target peptide ions. The fragmentation spectrum showed abundant c-/z- and a-/x-type ions, attributable to H attachment/abstraction to/from peptide ion. Because the low-energy neutral H radical initiated the fragmentation, the charge state of the precursor ion was maintained during the dissociation. As a result, precursor ions of any charge state, including singly charged positive and negative ions, could be analyzed for amino acid sequence. The sequence coverage exceeding 90% was obtained for both singly protonated and singly deprotonated substance P peptide. This mass spectrometry also preserved labile post-translational modification bonds. The modification sites of triply phosphorylated peptide (kinase domain of insulin receptor) were identified with the sequence coverage exceeding 80%.

Quantitative Matrix-assisted Laser Desorption/ionization Mass Spectrometry of Pyrene-derivatized Glycopeptides for Investigation of Mammalian Cell Glycomics

Glycan is one of the major information-rich biomolecules. Alterations in N-glycans can be associated with many diseases including cancer, and are often observed in the serum of affected patients. As an example, prostate-specific antigen (PSA) is a glycoprotein secreted by prostatic epithelial cells. The serum PSA assay is widely used for detection of prostate cancer (PCa). Detection of altered PSA glycan is regarded as a precise diagnosis. However, the limited amount of serum PSA available makes it difficult to determine detailed glycan structures, as the PSA level in serum is very low. Mass spectrometry (MS) is a powerful tool for analyzing glycan structures. We have recently established a highly sensitive MS for both glycans and glycopeptides by pyrene derivatization. Matrix-assisted laser desorption/ionizationquadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectra of glycans derivatized with pyrene butanoic acid hydrazide (PBH) were compared with those of pyridylaminated (PA) glycans. Each derivatized glycan was prepared from the same amount of commercial PSA. PBH-labeled PSA glycans showed higher signal intensity with less fragmentation compared with PA-glycans, and gave spectra with high s/n ratio (in both positive- and negative-ion modes). For derivatization of the glycopeptides, pyrenyl diazomethane (PDAM) was used. The glycopeptide signals were greatly enhanced by this derivatization. PDAM-glycopeptides prepared from about 10 ng of PSA could be determined. In this study, we demonstrated that MS analysis using pyrene derivatization provides higher sensitivity and stability for both glycans and glycopeptides, compared with HPLC and lectin affinity chromatography; furthermore comparable results were obtained to those seen with the other methods. We therefore conclude that our method is useful for investigation of mammalian cell glycomics.