Yuko Fukuyama

Ionic Liquid Matrixes Optimized for MALDI-MS of Sulfated/Sialylated/Neutral Oligosaccharides and Glycopeptides

1,1,3,3-tetramethylguanidium (TMG) salt of alpha-cyano-4-hydroxycinnamic acid (CHCA) (G(2)CHCA) was reported by Tatiana et al. as a useful ionic liquid matrix (ILM) for sulfated oligosaccharides to suppress the loss of sulfate groups. However, the report mainly referred to positive ion spectra only and amounts of 10 pmol or more of the analyte were used. Herein, we demonstrated highly sensitive detection of sulfated/sialylated/neutral oligosaccharides and preferential ionization of glycopeptides by optimizing a newly synthesized ILM: TMG salt of p-coumaric acid (G(3)CA) and the existing G(2)CHCA in both positive and negative ion extraction modes. Sulfated oligosaccharides were detected with high sensitivity (e.g., 1 fmol) in both ion extraction modes, and the dissociation of sulfate groups was suppressed especially using G(3)CA. Sialylated and neutral oligosaccharides were also detected with high sensitivity (e.g., 1 fmol) with positive ion extraction while the dissociation of sialic acids was suppressed especially using G(3)CA. Additionally, glycopeptide ions were detected preferentially using the ILMs among the digest of a glycoprotein, ribonuclease B, in both ion extraction modes but particularly in the negative ion mode. As a result, the use of optimized ILMs provides an effective method for carbohydrate analysis due to the highly sensitive soft-ionization achieved in both ion extraction modes as well as the homogeneity of analyte-matrix mixtures.

β‐Carboline alkaloids as matrices for UV‐matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry in positive and negative ion modes. Analysis of proteins of high molecular mass, and of cyclic and acyclic oligosaccharides

We report that commercially available beta-carbolines (nor-harmane (9H-pyrido[3,4-b]indole), harmane (1-methyl-9H-pyrido[3,4-b]indole), harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole), harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol), harmaline (3,4-dihydro-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) and harmalol (3,4-dihydro-1-methyl-9H-pyrido[3,4-b]indol-7-ol)), are useful MALDI matrices at 337 nm, for cyclic oligosaccharides (cyclodextrins, range 972-1290 Da), acyclic oligosaccharides (range 342-828 Da) and high molecular mass proteins (range 23,290-66,525 Da) in both positive and negative modes. This was investigated by using time-of-flight (TOF) mass spectrometers of different sensitivities, equipped with and without pulse extraction facilities. A comparison with conventional matrices for carbohydrates (DHB and DHB/HIC) indicates that beta-carbolines provide the same level of sensitivity and resolution in the positive mode, but offer the advantage of high levels of sensitivity and resolution in the negative mode. Harmaline has been found to be specially effective for the analysis of high-mass proteins in both modes, and also exhibits excellent experimental reproducibility of the results owing to the homogeneous crystallization of the analyte-matrix mixture over the entire sample surface area. Harmane and nor-harmane are both excellent matrices for high-mass proteins also. As MALDI matrices, beta-carbolines permit measurement of sulfated sugars in the negative ion mode as ([M-H]), and of neutral sugars and proteins as both [M + H]+ and [M-H]- in appropriate modes.

Highly sensitive MALDI analyses of glycans by a new aminoquinoline-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix.

In glycomics, mass spectrometry is an indispensable tool for high throughput analyses. Generally speaking, glycans contain many hydroxyl groups and are more difficult to ionize than peptides. Derivatization of glycans has been useful for increasing sensitivity. However, it takes time to purify and causes loss of sample. Here, we show a highly sensitive aminoquinoline (AQ)-labeling method of glycans on a matrix-assisted laser desorption/ionization (MALDI) target using a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA). It is a rapid procedure and reduces loss of sample material during the reaction process, especially in negative ion mode where 10 amol of monosialylated N-glycan were detected as AQ-labeled molecular ions. In addition, MS/MS of 10 amol of monosialylated N-glycan was achieved.

Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi Identification of sulfated high-mannose type oligosaccharides

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C‐terminal extension that contains a number of post‐translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV‐MALDI‐TOF mass spectrometry in conjunction with peptide N‐glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N‐linked oligosaccharides present in this domain. The UV‐MALDI‐TOF MS analysis in the negative‐ion mode, using nor‐harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high‐mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV‐MALDI‐TOF MS analysis (a) a main population of high‐mannose type oligosaccharides was shown in the positive‐ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.

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.

Matrix-assisted ultraviolet laser-desorption ionization and electrospray-ionization time-of-flight mass spectrometry of sulfated neocarrabiose oligosaccharides.

Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS). UV-MALDI-TOF-MS was carried out in the linear and reflectron modes and, as routine, in both the positive- and negative-ion modes. 2,5-Dihydroxybenzoic acid and nor-harmane were used as matrices. In the positive- and negative-ion modes, with both matrices, peaks corresponding to (M+Na)(+) and (M-Na)(-) ions, respectively, were obtained, with only some signals due to glycosidic linkage cleavages (prompt fragmentation). With 2,5-dihydroxybenzoic acid abundant matrix signals were observed; nor-harmane afforded very few matrix signals in both ion modes, but more desulfation (prompt fragmentation) of the compounds occurred. When the desorption/ionization process was highly efficient, the post-source decay (PSD) fragmentation patterns were also investigated; most of the fragments detected derived from glycosidic linkage cleavages. Electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) in the negative-ion mode confirmed, with the observation of the (M-Na)(-) and the multiply charged anions, the identity and the purity of the samples.

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.

Rapid quantitative profiling of N-glycan by the glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.

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.

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.