p-Coumaric Acid as a MALDI Matrix for

Feb 5, 2014 - A cool and high salt-tolerant ionic liquid matrix for preferential ionization of phosphopeptides by negative ion MALDI-MS. Ling Ling , C...
0 downloads 9 Views 996KB Size
Download PDF
Technical Note pubs.acs.org/ac

3‑Aminoquinoline/p‑Coumaric Acid as a MALDI Matrix for Glycopeptides, Carbohydrates, and Phosphopeptides Yuko Fukuyama,* Natsumi Funakoshi, Kohei Takeyama, Yusaku Hioki, Takashi Nishikaze, Kaoru Kaneshiro, Shin-ichirou Kawabata, Shinichi Iwamoto, and Koichi Tanaka Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan S Supporting Information *

ABSTRACT: 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-cyano4-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 3AQ/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.

G

Recently, liquid matrices that maintain their liquid states in a vacuum have been reported to improve sensitivity and reproducibility for biomolecules compared with solid matrices.10−28 3-Aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA), introduced by Kolli et al. in 1996,10 has been reported to be useful for biopolymers.10−17 It has been reported to increase sensitivity for carbohydrates13−15 or phosphopeptides,17 but it has not been systematically evaluated for glycopeptides. The sensitivity improvement with 3-AQ/ CHCA is probably due to its property separating or concentrating hydrophilic carbohydrates,16 glycopeptides,16 and phosphopeptides17 in a small central area in the matrix droplet, in addition to the ionization property of the matrix itself. However, 3-AQ/CHCA has a problem as a “hot” matrix that enhances the dissociation of labile regions. 1,1,3,3Tetramethylguanidium (TMG, G) salt of p-coumaric acid (CA) (G3CA) as a liquid matrix has been reported to improve sensitivity of carbohydrates and suppress dissociation of sulfate

lycosylation and phosphorylation are important posttranslational modifications (PTMs) in biological processes and biomarker research.1−6 Glycosylation is found in many cancer diagnostic markers, and glycosylation patterns or a specific glycoform are key elements in the differentiation and progression of diseases.1−4 Phosphorylation is a transient modification related to signaling pathways that acts as a regulator of protein activities or cellular pathways.1,5,6 Hence, detection of glycosylation or phosphorylation is significant for biomarker discovery but is a challenging task due to their low abundance.1−6 Matrix-assisted laser desorption/ionization (MALDI)7,8 and electrospray ionization9 mass spectrometry (MS) are indispensable analytical tools for proteomics but remain under development for PTM analyses. The difficulty in PTM analyses is mainly sensitivity and the dissociation of labile regions such as sialic acids and phosphate groups. One solution in MALDIMS is to improve matrices to address these issues. 2,5Dihydroxybenzoic acid (2,5-DHB), a hydrophilic “cool” matrix, has been conventionally used for the analytes to resolve the issues. © 2014 American Chemical Society

Received: November 15, 2013 Accepted: January 27, 2014 Published: February 5, 2014 1937

dx.doi.org/10.1021/ac4037087 | Anal. Chem. 2014, 86, 1937−1942

Analytical Chemistry

Technical Note

Table 1. Glycopeptides, Carbohydrates, and Phosphopeptides Used in This Study

from fetal calf serum, angiotensin II, 1,4-dithiothreitol (DTT), iodoacetoamide (IAA), cellulose fibrous medium, and ammonium dihydrogen phosphate (ADP) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). p-Coumaric acid (CA) was custom-ordered in purified form from Dojindo Laboratories (Kumamoto, Japan) or NARD Institute, Ltd. (Osaka, Japan). The glycopeptide, carbohydrate, and phosphopeptide standards used in this study are listed in Table 1. Glycopeptides (analyte 1 to 10 in Table 1) were prepared as described later. Carbohydrates (analyte 11 to 14 in Table 1) were obtained from Takara Bio Inc. (Shiga, Japan). Trypsin (gold, mass spectrometry grade) was from Promega Corp. (Madison, WI, USA). Sialidase A was purchased from Prozyme, Inc. (Hayward, CA, USA). Acetonitrile (CH3CN) and trifluoroacetic acid (TFA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Water used in all experiments was deionized using a Milli-Q ultrapure water purification system (Merck, Ltd., Tokyo, Japan).

groups and sialic acids compared with TMG salt of CHCA (G2CHCA) as a liquid matrix or 2,5-DHB.23−26 The better suppression effect of G3CA25 compared with G2CHCA24 probably depends on the difference between CA and CHCA. Herein, a novel matrix 3-AQ/CA consisting of 3-AQ and CA is introduced for glycopeptides, carbohydrates, and phosphopeptides. CA, which is hydrophilic, is expected to have a higher affinity for hydrophilic compounds than CHCA and to suppress dissociation of labile regions like G3CA. The sensitivity and suppression of dissociation of labile regions are evaluated using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-DHB.

■

EXPERIMENTAL SECTION Materials. α-Cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (2,5-DHB) were purchased from LaserBio laboratories (Sophia-Antipois Technopole, France). 3Aminoquinoline (3-AQ), β-casein 33-48, β-casein 1−25, transferrin human, fetuin from fetal calf serum, asialofetuin 1938

dx.doi.org/10.1021/ac4037087 | Anal. Chem. 2014, 86, 1937−1942

Analytical Chemistry

Technical Note

Preparation of Glycopeptide Standards. Transferrin, fetuin, or asialofetuin (100 nmol) was dissolved in urea (8 M) in Tris-HCl buffer (0.1 M, pH 8.7). DTT (1 M) was added (final concentration of 10 mM) and incubated for 1 h at 56 °C; then, IAA (1 M) was added (final concentration of 100 mM) and incubated for 45 min at room temperature. After cooling, the obtained solution was desalted with water and concentrated with Tris-HCl buffer (0.1 M, pH 8.7). Following the addition of trypsin dissolved in Tris-HCl buffer (0.1 M, pH 8.7) for transferrin, fetuin, and asialofetuin at an enzyme/substrate ratio of 1:100 (w/w), the obtained mixtures were incubated at 37 °C overnight. Glycopeptides were enriched by cellulose fibrous medium.29 They were detached by CH3CN/H2O/TFA (80:20:0.1, v/v) and eluted by CH 3 CN/H 2 O/TFA (50:50:0.1, v/v).30,31 The aliquots were dried in a vacuum centrifuge and refrigerated. A glycopeptide-enriched sample was fractionated using the Nexera ultrahigh-performance liquid chromatography system (Shimadzu Corporation, Japan) with a Shim-Pack FC-ODS 4.6 × 150 mm column (Shimadzu Corporation, Japan). Cellulose-processed samples were dissolved in CH3CN/H2O/TFA (5:95:0.1, v/v). Transferrin, fetuin, and asialofetuin glycopeptides were isolated by a lineargradient using CH3CN/H2O/TFA (5:95:0.05, v/v) as solvent A and CH3CN/H2O/TFA (90:10:0.05, v/v) as solvent B at a flow rate of 1 mL/min. The collected fractions were dried and refrigerated. Asialotransferrin GP1 or GP2 was obtained by removing sialic acids from the collected transferrin GP1 or GP2. The collected transferrin GP1 or GP2 was dissolved in water and mixed with Sialidase A in Tris-HCl buffer (20 mM, pH 7.5) and phosphate buffer (pH 6.0) and then incubated at 37 °C for 60 min. The obtained solution was dried and refrigerated. Quantification of the glycopeptides was determined by a calibration curve method using angiotensin II as a external standard (R2 = 0.999), which was corrected with molar extinction coefficients using the UV absorption of the constituent amino acids.31,32 The quantified glycopeptides (analyte 1 to 10 in Table 1) were used as analytes for MALDI-MS. Matrices. 3-AQ/CA (Figure 1) was prepared as follows. 3AQ or CA was dissolved in CH3CN/H2O (50:50, v/v) with

(50:50, v/v) or CH3CN/H2O/TFA (50:50:0.1, v/v) at 1 mg/ mL or 5 mg/mL. Sample Preparation for Glycopeptides. Glycopeptides (analytes 1 to 10 in Table 1) were dissolved in water at appropriate concentrations by 10-fold serial dilutions. 3-AQ/ CA or 3-AQ/CHCA was prepared by dissolving in CH3CN/ H2O (50:50, v/v) with ADP (2 mM) as described above. 2,5DHB was prepared at 5 mg/mL as described above. Matrix solution and analyte solution were mixed in a small plastic tube at 1:1 (v/v), and the obtained mixture (0.5 μL) was dropped on a μFocus 700 μm MALDI plate (Hudson Surface Technology, Inc., NJ, USA). Sample Preparation for Carbohydrates. Carbohydrates (analytes 11 to 14 in Table 1) were dissolved in water at appropriate concentrations by 10-fold serial dilutions. 3-AQ/ CA or 3-AQ/CHCA was prepared by dissolving in CH3CN/ H2O (50:50, v/v) with ADP (2 mM) as described above. 2,5DHB was prepared at 1 mg/mL as described above. The matrix solution and analyte solution were mixed in a small plastic tube at 1:1 (v/v), and the obtained mixture (1 μL) was dropped on a μFocus 700 μm MALDI plate. Sample Preparation for Phosphopeptides. Phosphopeptides (analytes 15 and 16 in Table 1) were dissolved in CH3CN/H2O (50:50, v/v) or CH3CN/H2O/TFA (50:50:0.1, v/v) at appropriate concentrations by 10-fold serial dilutions. 3AQ/CA or 3-AQ/CHCA was prepared by dissolving in CH3CN/H2O (50:50, v/v) with ADP (20 mM) as described above. 2,5-DHB was prepared at 5 mg/mL as described above. Matrix solution (0.5 μL) and analyte solution (0.5 μL) were deposited on a μFocus 700 μm MALDI plate. MALDI-MS. A MALDI-quadrupole ion trap (QIT) time-offlight mass spectrometer (TOFMS) measurement was performed using an AXIMA Resonance (Shimadzu/Kratos, UK) mass spectrometer equipped with a nitrogen UV laser (337 nm) in both positive and negative ion modes. The detection limit was defined as the lowest quantity of analytes detected as [M + H]+ or [M − H]− with a signal-to-noise ratio (S/N) exceeding 2 within an error ratio of 25%, wherein each analyte solution was evaluated at different concentrations made by 10-fold serial dilutions.

■

RESULTS AND DISCUSSION Optimization of 3-AQ/CA. 3-AQ/CA was optimized for transferrin GP1 (analyte 1 in Table 1). For 3-AQ and CA mixed at 1:1 to 20:1 (mol/mol), the highest sensitivity was obtained at 9:1 (mol/mol). This was the same ratio as 3-AQ/ CHCA (see the Experimental Section). ADP added to 3-AQ/ CA improved the sensitivity 10-fold, which corresponds to the result of 3-AQ/CHCA for phosphopeptides.17 Compared with 3-AQ/CA, known liquid matrices,10,19,24,25 and 2,5-DHB, the sensitivity was higher using 3-AQ/CA or 3-AQ/CHCA, and dissociation of sialic acids was suppressed more using 3-AQ/ CA. The optimized 3-AQ/CA was used in all the experiments described below. Glycopeptide Analyses Using 3-AQ/CA. The sensitivity for sialoglycopeptides and asialoglycopeptides from transferrin or fetuin (analytes 1 to 10 in Table 1) was evaluated using 3AQ/CA, 3-AQ/CHCA, and 2,5-DHB. The detection limits for [M + H]+ or [M − H]− are indicated in Table 2. This was improved 1- to 10-fold for sialoglycopeptides (analytes 1 to 5 in Table 2) using 3-AQ/CA compared with 3-AQ/CHCA or 2,5DHB and approximately 1- to 10-fold for asialoglycopeptides (analytes 6 to 10 in Table 2) using 3-AQ/CA or 3-AQ/CHCA

Figure 1. 3-Aminoquinoline (3-AQ)/p-coumaric acid (CA) (3-AQ/ CA) as a novel liquid matrix.

ADP (2 mM or 20 mM) at 100 nmol/μL. 3-AQ solution and CA solution were mixed at 9:1 (v/v) (3-AQ:CA at 9:1, mol/ mol) to be used as a matrix. Alternatively, 3-AQ (20 mg) was dissolved in CA solution (150 μL) in which CA (10 mg) was dissolved in CH3CN/H2O (50:50, v/v, 600 μL) with ADP (2 mM). The obtained mixture was diluted ten times with CH3CN/H2O (50:50, v/v) with ADP (2 mM) to be used as a matrix. 3-AQ/CHCA was prepared as follows. 3-AQ (20 mg) was dissolved in CHCA solution (150 μL) in which CHCA (10 mg) was dissolved in CH3CN/H2O (50:50 (v/v), 600 μL) with ADP (2 mM). The obtained mixture was diluted ten times with CH3CN/H2O (50:50, v/v) with ADP (2 mM) to be used as a matrix. 2,5-DHB was prepared by dissolving in CH3CN/H2O 1939

dx.doi.org/10.1021/ac4037087 | Anal. Chem. 2014, 86, 1937−1942

Analytical Chemistry

Technical Note

Table 2. Detection Limits of Glycopeptides, Carbohydrates, and Phosphopeptides Using 3-AQ/CA, 3-AQ/CHCA, and 2,5DHBa detection limit (f mol/well) [M − H]−

[M + H]+ no.

name

Glycopeptides Sialoglycopeptides 1 transferrin GP1 2 transferrin GP2 3 fetuin GP1 4 fetuin GP2 5 fetuin GP3 Asialoglycopeptides 6 asialotransferrin GP1 7 asialotransferrin GP2 8 asialofetuin GP1 9 asialofetuin GP2 10 asialofetuin GP3 Carbohydrates (PA-Labeled Glycans) Sialoglycans 11 A1 glycan 12 A2 glycan Neutral Glycans 13 NA2 glycan 14 NA4 glycan Phosphopeptides 15 β-casein 33−48 16 β-casein 1−25

3-AQ/CA

3-AQ/CHCA

2,5-DHB

3-AQ/CA

3-AQ/CHCA

2,5-DHB

10 10 10 100 1000

10 100 100 100 −

100 100 100 1000 −

1 10 10 10 100

1 10 10 100 1000

10 100 100 100 −

1 1 0.1 0.1 1

1 1 0.1 0.1 1

1 1 0.1 0.1 10

1 1 0.1 0.1 0.1

1 1 0.1 0.1 1

1 10 1 1 10

1 1

1 1

100 100

0.1 1

0.1 1

100 100

0.1 1

1 1

1 10

1 1

1 1

10 10

1 1

1 1

1 10

1 1

1 1

1 10

a

Detection limit for glycopeptides, carbohydrates, and phosphopeptides using 3-AQ/CA, 3-AQ/CHCA, and 2,5-DHB in positive- and negative-ion modes (see the Experimental Section). The detection limit was defined as the lowest quantity of analyte detected as [M + H]+ or [M − H]− with S/ N > 2 within an error ratio of 25% at different concentrations made by 10-fold serial dilutions. “−” indicates that no ions were detected for that analyte at ≤1000 fmol.

tion of sialic acids was suppressed using 3-AQ/CA in both positive- and negative-ion modes compared with 3-AQ/CHCA or 2,5-DHB (Figure S-2 in the Supporting Information). Figure 2 presents mass spectra of NA4 glycan (analyte 14 in Table 1) using 3-AQ/CA, 3-AQ/CHCA, or 2,5-DHB. Fragmentation of neutral carbohydrates was suppressed using 3-AQ/CA (Figure 2d), whereas it was observed using 3-AQ/CHCA (Figure 2e). All the sialylated and neutral glycans were analyzed with higher sensitivity and suppressed dissociation of sialic acids and fragmentation of neutral carbohydrates using 3-AQ/CA. Moreover, on-target 3-AQ labeling reported using 3-AQ/ CHCA13 was also possible using 3-AQ/CA for native carbohydrates. Our on-target 3-AQ labeling method enables us to detect neutral carbohydrates as phosphate adducts, which offer significant advantages in structural analysis by MS/MS.15 However, anionic adducts of neutral carbohydrates are known to be somewhat unstable, thereby causing non-negligible fragmentation in MS1 measurements. The “cool” nature of 3AQ/CA greatly suppresses the MS1 fragmentation compared to 3-AQ/CHCA, simplifying spectral interpretation (Figure S-3 in the Supporting Information). Phosphopeptide Analyses Using 3-AQ/CA. The sensitivity of mono- and tetraphosphopeptides from β-casein (analytes 15 and 16 in Table 1) was evaluated using 3-AQ/ CA, 3-AQ/CHCA, and 2,5-DHB. Table 2 indicates the detection limits for [M + H]+ or [M − H]− of mono- and tetraphosphopeptides. Detection limits were improved 1- to 10fold using 3-AQ/CA or 3-AQ/CHCA compared with 2,5-DHB, wherein the S/N for ion signals of [M + H]+ or [M − H]− was

rather than 2,5-DHB. The detection limit was improved for sialoglycopeptides more than for asialoglycopeptides, probably due to the dissociation of sialic acids (N-acetylneuramic acid; NANA). Dissociation of sialic acids for all sialoglycopeptides (analytes 1 to 5 in Table 2) was suppressed more using 3-AQ/ CA compared with 3-AQ/CHCA or 2,5-DHB in both positive and negative ion modes. Dissociation was most suppressed in negative-ion mode using 3-AQ/CA (Figure S-1 in the Supporting Information). Figure 2 presents mass spectra of fetuin GP1 (analyte 3 in Table 1) using 3-AQ/CA, 3-AQ/ CHCA, and 2,5-DHB. [M + H]+ of fetuin GP1 was most clearly detected using 3-AQ/CA (Figure 2a) compared with 3AQ/CHCA or 2,5-DHB (Figure 2b,c). [M − 3NANA + H]+ was detected with higher intensity than [M + H]+, [M − NANA + H]+, or [M − 2NANA + H]+ using 3-AQ/CHCA compared with 3-AQ/CA or 2,5-DHB. [M − H]− was most clearly observed using 3-AQ/CA (Figure S-1 in the Supporting Information). High-sensitivity analyses of glycopeptides with suppression of dissociation for labile regions were thus achieved by using 3-AQ/CA. Carbohydrate Analyses Using 3-AQ/CA. Sensitivity for sialylated and neutral PA-labeled glycans (analytes 11 to 14 in Table 1) was evaluated using 3-AQ/CA, 3-AQ/CHCA, and 2,5-DHB. The detection limits for [M + H]+ or [M − H]− are indicated in Table 2. For sialoglycans, this was improved 10- to 1000-fold using 3-AQ/CA or 3-AQ/CHCA compared with 2,5DHB. For neutral glycans, it was improved 10-fold using 3-AQ/ CA compared with 2,5-DHB. The detection limits using 3-AQ/ CA are comparable with those using 3-AQ/CHCA. Dissocia1940

dx.doi.org/10.1021/ac4037087 | Anal. Chem. 2014, 86, 1937−1942

Analytical Chemistry

Technical Note

Figure 2. Positive-ion mass spectra of 100 fmol fetuin GP1 (analyte 3 in Table 1), 100 fmol NA4 glycan (analyte 14 in Table 1), and 10 fmol βcasein 1−25 (analyte 16 in Table 1) using (a, d, g) 3-AQ/CA, (b, e, h) 3-AQ/CHCA, and (c, f, i) 2,5-DHB.

resulted in high-sensitivity analyses. In addition, the suppression of dissociation for labile regions with 3-AQ/CA is probably explained by the fact that 3-AQ/CHCA consists of CHCA, which as a “hot” matrix facilitates fragmentation and dissociation, whereas 3-AQ/CA consists of CA, which nearly does not ionize25 and has an affinity for hydrophilic compounds, forming a “cool” liquid matrix like G3CA,25 which is reported to suppress dissociation of labile regions of carbohydrates better than G2CHCA.24,25

higher using 3-AQ/CA rather than 3-AQ/CHCA. Dissociation of phosphate groups was suppressed more using 3-AQ/CA than 3-AQ/CHCA in both positive- and negative-ion modes. Figure 2 presents mass spectra of tetraphosphopeptide β-casein 1−25 (analyte 16 in Table 1) using 3-AQ/CA, 3-AQ/CHCA, and 2,5-DHB. Dissociation of four phosphate groups was suppressed using 3-AQ/CA (Figure 2g) compared with using 3-AQ/CHCA (Figure 2h). The dissociation was enhanced more using 3-AQ/CHCA compared with 3-AQ/CA or 2,5DHB. Phosphopeptides were thus analyzed with higher sensitivity, and the dissociation of phosphate groups was suppressed more using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-DHB. Sensitivity Improvement and Suppression of Dissociation Using 3-AQ/CA. It was confirmed that 3-AQ/CA improved the sensitivity of glycopeptides, carbohydrates, and phosphopeptides (Table 2) and that it suppressed the dissociation of sialic acids or phosphate groups and the fragmentation of neutral carbohydrates (Figure 2) better than 3-AQ/CHCA or 2,5-DHB. The sensitivity improvement by 3AQ/CA can be explained by its difference from 3-AQ/CHCA, that is, the difference between CHCA and CA as a counterion. CHCA is known to have high ionization property as a versatile matrix and is known to be hydrophobic, unlike CA. However, CA has difficulty ionizing analytes25 and is rather hydrophilic. Hydrophilic compounds such as glycopeptides, carbohydrates, and phosphopeptides have higher affinity for 3-AQ/CA than for 3-AQ/CHCA. Thus, the analytes are concentrated in a small area of an analyte/matrix droplet using 3-AQ/CA, as much or more as reported for 3-AQ/CHCA,16,17 which

■

CONCLUSION

■

ASSOCIATED CONTENT

This study introduced 3-AQ/CA as a matrix for glycopeptides, carbohydrates, and phosphopeptides. 3-AQ/CA improved the sensitivity to a maximum of 1000-fold, especially compared with using 2,5-DHB, and suppressed the dissociation of sialic acids or phosphate groups and fragmentation of neutral carbohydrates compared with using 3-AQ/CHCA or 2,5DHB. Thus, 3-AQ/CA resolved the issues with 3-AQ/CHCA as a “hot” matrix. Consequently, a liquid matrix 3-AQ/CA is expected to facilitate glycosylation and phosphorylation analyses in MALDI-MS.

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 1941

dx.doi.org/10.1021/ac4037087 | Anal. Chem. 2014, 86, 1937−1942

Analytical Chemistry

■

Technical Note

(28) Schnöll-Bitai, I.; Ullmer, R.; Hrebicek, T.; Rizzi, A.; Lacik, I. Rapid Commun. Mass Spectrom. 2008, 22, 2961−2970. (29) Wada, Y.; Tajiri, M.; Yoshida, S. Anal. Chem. 2004, 76, 6560− 6565. (30) Yu, L.; Li, X.; Guo, Z.; Zhang, X.; Liang, X. Chem.Eur. J. 2009, 15, 12618−12626. (31) Mysling, S.; Palmisano, G.; Højrup, P.; Thaysen-Andersen, M. Anal. Chem. 2010, 82, 5598−5609. (32) Kuipers, B. J. H.; Gruppen, H. J. Agric. Food Chem. 2007, 55, 5445−5451.

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-75-823-2897. Fax: +81-75-823-2900. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST Program)”, initiated by the Council for Science and Technology Policy (CSTP). The authors thank Mrs. Azusa Yagi and Mr. Takashi Ibuki for technical support.

■

REFERENCES

(1) Shukla, H. D.; Vaitiekunas, P.; Cotter, R. J. Proteomics 2012, 12, 3085−3104. (2) Morelle, W.; Canis, K.; Chirat, F.; Faid, V.; Michalski, J.-C. Proteomics 2006, 6, 3993−4015. (3) Blom, N.; Sicheritz-Pontén, T.; Gupta, R.; Gammeltoft, S.; Brunak, S. Proteomics 2004, 4, 1633−1649. (4) Zhang, X.; Wei, D.; Yap, Y.; Li, L.; Guo, S.; Chen, F. Mass Spectrom. Rev. 2007, 26, 403−431. (5) Reinders, J.; Sickmann, A. Proteomics 2005, 5, 4052−4061. (6) Thingholm, T. E.; Jensen, O. N.; Larsen, M. R. Proteomics 2009, 9, 1451−1468. (7) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151−153. (8) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299−2301. (9) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64−71. (10) Kolli, V. S. K.; Orlando, R. Rapid Commun. Mass Spectrom. 1996, 10, 923−926. (11) Sze, E. T. P.; Chen, T.-W. D.; Wang, G. J. Am. Soc. Mass Spectrom. 1998, 9, 166−174. (12) Yang, S.-H.; Reddy, P. M.; Ho, Y.-P. Anal. Chem. 2010, 82, 44− 48. (13) Kaneshiro, K.; Fukuyama, Y.; Iwamoto, S.; Sekiya, S.; Tanaka, K. Anal. Chem. 2011, 83, 3663−3667. (14) Kaneshiro, K.; Watanabe, M.; Terasawa, K.; Uchimura, H.; Fukuyama, Y.; Iwamoto, S.; Sato, T.-A.; Shimizu, K.; Tsujimoto, G.; Tanaka, K. Anal. Chem. 2012, 84, 7146−7151. (15) Nishikaze, T.; Kaneshiro, K.; Kawabata, S.; Tanaka, K. Anal. Chem. 2012, 84, 9453−9461. (16) Sekiya, S.; Taniguchi, K.; Tanaka, K. Rapid Commun. Mass Spectrom. 2012, 26, 693−700. (17) Fukuyama, Y.; Takeyama, K.; Kawabata, S.; Iwamoto, S.; Tanaka, K. Rapid Commun. Mass Spectrom. 2012, 26, 2454−2460. (18) Armstrong, D. W.; Zhang, L.-K.; He, L.; Gross, M. L. Anal. Chem. 2001, 73, 3679−3686. (19) Mank, M.; Stahl, B.; Boehm, G. Anal. Chem. 2004, 76, 2938− 2950. (20) Tholey, A. Rapid Commun. Mass Spectrom. 2006, 20, 1761− 1768. (21) Tholey, A.; Heinzle, E. Anal. Bioanal. Chem. 2006, 386, 24−37. (22) Palmblad, M.; Cramer, R. J. Am. Soc. Mass Spectrom. 2007, 18, 693−697. (23) Laremore, T. N.; Murugesan, S.; Park, T.-J.; Avci, F. Y.; Zagorevski, D. V.; Linhardt, R. J. Anal. Chem. 2006, 78, 1774−1779. (24) Laremore, T. N.; Zhang, F.; Linhardt, R. J. Anal. Chem. 2007, 79, 1604−1610. (25) Fukuyama, Y.; Nakaya, S.; Yamazaki, Y.; Tanaka, K. Anal. Chem. 2008, 80, 2171−2179. (26) Nishikaze, T.; Fukuyama, Y.; Kawabata, S.; Tanaka, K. Anal. Chem. 2012, 84, 6097−6103. (27) Ulmer, R.; Rizzi, A. M. J. Mass Spectrom. 2009, 44, 1596−1603. 1942

dx.doi.org/10.1021/ac4037087 | Anal. Chem. 2014, 86, 1937−1942