アルギニン残基修飾/除去によるMALDI-MS/MS解析における糖ペプチドのフラグメンテーションの改善 Improving Fragmentation of Arginine-Containing Glycopeptide Through Modifying or Removing Arginine Residue in MALDI Mass Spectrometry-Based Sequence Analysis

MSSJ2013 1P - 02

○谷口謙一 ・九山浩樹 ・梶原茂樹 ・田中耕一 株式会社島津製作所 田中最先端研究所

○ Kenichi Taniguchi, Hiroki Kuyama, Shigeki Kajihara, Koichi Tanaka Koichi Tanaka Laboratory of Advanced Science and Technology (KTLAST), Shimadzu Corporation

3. RESULTS and DISCUSSIONS 1. INTRODUCTION

2. EXPERIMENTAL

4.CONCLUSIONS

Material

Glycopeptide Purification

Glycopeptide Derivatization

RP-HPLC Separation and Mass Spectrometry

Peak Processing

MS/MS Ion Search

(a)

Score 28(29)

: N-acetylglucosamine

y 6

y 7

y 8

y 13

【M+H】+

X5

y ４

y 5

y 9

y 10

y 11

500 1000 1500 2000 2500 m/z

b 3

b 5

b 6

b 7

b 8

b 9

b 10

b 11

b 13

500 1000 1500 2000 2500 m/z

b 4

Score 55(62)

【M+H】+

(b)

Fig.2 Schematic depiction of enzymatic removal or modification of Arg residue.

Fig.5 Peptide sequencing of T4-1 and G5-1. The effect of Arg removal or modification and tertiary amine labeling on MS3 fragmentation is presented. MS3 spectrum of (a) intact T4-1, (b)labeled and Arg-removed T4-1, (c) intact G5-1 and (d) labeled and Arg-modified T4-1. The score in parentheses indicates the value obtained after neutral-loss filtering of 203Da peaks.

Mascot score comparison of glycopeptides from trypsin or GluC digest of human α1-acid glycoprotein. The entries from T4-1 to G5-1 are Arg-containing glycopeptides; entries from T1-1 to T3 are glycopeptides containing lysine instead of Arg residue. The Mascot scores are the average of three experiments. Error bars represent standard deviation of the mean. Elevated levels were calculated as: “Score (TMT + Arg removal/modification + Filter (203)) - Score (No Label)” in Arg-containing glycopeptides and “Score (TMT + Filter (203)) - Score (No Label)” in lysine-containing glycopeptides.

Fig.6

Fig.3 LC-MALDI-MS spectrum of derivatized glycopeptides derived from trypsin digestion of human α1 acid glycoprotein using Mass++.

Fig.1 Deteriorated fragmentation of Arg-containing glycopeptide and its potential solution

Acknowledgements This research is supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)."

References K.Taniguchi, H.Kuyama, S.Kajihara, K.Tanaka, J. Mass Spectrom., 2013, 48, 951–960.

The digest solutions derived from Trypsin or GluC digestion of α1-acid glycoprotein were subjected to size exclusion chromatography using a 2.1mm×220mm Superdex Peptide column (GE Healthcare, Uppsala, Sweden) and a NexeraTM high-performance liquid chromatography (HPLC) system (Shimadzu, Japan), with 2mM NaHCO3 as an eluent at a flow rate of 150µl/min. Fractions were collected for 5 to 7min. Under this condition, the N-linked glycopeptides were well separated from the non-glycosylated peptides.

Each dried glycopeptide fraction was dissolved in 19 µl of 80% acetonitrile/water 0.4M 4-methylmorpholine. A volume of 1.5 µl of 57mM N-succinimidyl 4-aza-6-(2,6-dimethyl-1-piperidinyl)-5-oxohexanoate (in acetonitrile) was then added to the solution. The mixture was incubated at room temperature for 30min, and 50 µl of water was added to stop the reaction. After the evaporation, the amino group-derivatized glycopeptides were dissolved in 19 µl of 100mM NaHCO3 solution. Carboxypeptidase B solution ( 1µl, 5.7µM) was added, and the resulting solution was incubated at 37 °C for 2 h. For citrullination, the glycopeptides were dissolved in 20 µl of 50mM Tris–HCl buffer (pH 7.4) containing 10mM CaCl2 and 2 mM DTT. Five microliters of PAD4 was added to this solution and incubated at 37 ℃ for overnight.

The amino group-derivatized and Arg-removed glycopeptides were separated by RP chromatography on a NexeraTM system (Shimadzu, Japan). A C18 RP-HPLC column (Shim-Pack FC-ODS, 2.0mm ×150mm, Shimadzu, Japan) was used for the separation at a flow rate of 200 µl/min with a linear gradient profile of 0 to 60% solvent B over 35min. Solvent A was water containing 0.1% (v/v) acetonitrile and 0.05% (v/v) TFA, and solvent B was 80% acetonitrile containing 0.05% (v/v) TFA. The fraction was spotted onto a DHB and MDPNA[2]-precoated u-focus MALDI target plate (700 um diameter) which was purchased from Hudson Surface Technology, Inc. (Old Tappan, NJ, USA). The mass spectra were acquired using an AXIMA PerformanceTM mass spectrometer and MS/MS spectra were obtained using an AXIMA ResonanceTM UV-MALDI mass spectrometer (Shimadzu/Kratos, UK).

The peak lists for the database search were generated from MS spectra using Mass++ (version 2.3.0). The peaks in the MS spectrum were detected using the advanced peak-picking function MWD in Mass++. The neutral-loss peaks were then removed by the peak-filtering function that was originally developed in Mass++. Mass++ can be freely downloaded from the website http://www.first-ms3d.jp/english/.

MS/MS ion searches were performed by Mascot (version 2.4.0, Matrix Science, UK) on a local server with some customization. SwissProt (released January 11, 2011) was used as the database, and the taxonomy was set to Homo sapiens (human). Trypsin, Trypsin + CarboxypeptidaseB-R (customized), or None was selected as the enzyme, with no potential cleavage. The mass tolerances of peptide and fragment ion were 0.3 Da. Fixed modifications were carboxamidomethylation of cysteine (+57 Da), citrullination of Arg (+1 Da, customized) and lysine TMT labeling (+224 Da). The variable modifications were HexNAc (+203 Da), N-terminal TMT labeling (+224 Da) and N-terminal glutamine deamination (-17 Da). MALDI-QIT-TOF was selected as the instrument type.

High-purity MS grade 2,5-dihydroxybenzoic acid (DHB) was purchased from Shimadzu GLC (Tokyo, Japan). Human α1-acid glycoprotein, GluC and methylenediphosphonic acid (MDPNA) were purchased from Sigma (St. Louis, MO, USA). Sequencing-grade modified trypsin was obtained from Promega (Madison, WI, USA). Carboxypeptidase B was purchased from Worthington Biomedical Corp. (Lakewood, CO, USA). N-Succinimidyl 4-aza-6-(2,6-dimethyl-1- piperidinyl)-5-oxohexanoate (Tandem Mass Tag, (TMT)) was purchased from Thermo Fisher Scientific (San Jose, CA, USA). PAD4 (human recombinant) was purchased from Cayman Chemical Company (Lakewood, CO, USA)

Removal or modification of the Arg residue, tertiary amine labeling and neutral-loss filtering are useful for analyzing glycan structures, identifying glycoproteins and determining N-glycan binding positions.

Glycosylation is one of the most abundant post-translational modifications, and more than 50% of human protein is estimated to be glycosylated. Profiling glycoprotein requires the structural characterization of both glycan and peptide moieties, and the clarification of glycan modification position. Arginine (Arg)-containing peptides exhibit a low degree of structurally informative fragmentation when investigated using the commonly used collision-induced dissociation (CID) analysis. This is also true with glycopeptides. Here we present an approach for enhancing the structurally informative fragmentation of Arg-containing glycopeptides[1].

[ 1 ]

Fig.4 Glycan sequencing of T4-1 and G5-1. The effect of Arg removal or modification and tertiary amine labeling on MS/MS fragmentation is presented. MS/MS spectrum of (a) intact T4-1, (b) labeled and Arg-removed T4-1, (c) intact G5-1 and (d) labeled and Arg-modified G5-1.

MS3

Less informative fragmentation

Solution

R NH

HN NH2 H+

D

MS/MS Strong basicity

Proton is captured

Inhibit the cleavage of glycosidic bond

H.Kuyama, K.Sonomura, O,Nishimura, Rapid Commun. Mass Spectrom., 2008, 22, 1109–1116.

[ 2 ]

0 10 20 30 40 50 60 70 80 90

No Label No Label + Filter (203) TMT Label TMT + Filter (203) TMT+ Arg removal TMT+ Arg removal +Filter (203)

Sequence

T1-1*

T2

T3

T4-1

T5-1

T1-2*

T4-2

T5-2

T1-1

Entry

T1-2

Pyr-IPLCANLVPVPITN ATLDQ ITGK

LVPVPITNATLDQ ITGK

NEEYNK

SVQEIQATFFYFTPNK

QDQCIYNTTYLNVQR

ENGTISR

Pyr-IPLCANLV PVPITNATLDR

LVPVPITNATLDR

QNQCFYNSSYLNVQR

ENGTVSR

34

30

27

20

Elevated level

14

7

53

9

4

-

G5-1 NGTISRYVGGQE 6

TMT+ Arg modification TMT+ Arg modification + Filter (43,203)

300 600 900 1200 1500 1800 m/z

y 4

X10

y 5

y 6

y 8

y 9

y 10

y 11

(c)

Score 37(40)

b 1

b 2

b 3

b 4

b 5

b 6

b 7

b 8

b 9

b 11

300 600 900 1200 1500 1800 m/z

X10 X2 X5

(d)

Score 28(43)

【M+H】+

R NH

HN NH2 H+

D

MS/MS

proton is mobile Remove

Arg residue

H+

Add appropriately basic group

Induce the cleavage of glycosidic bond

R NH

HN NH2

D base H+ R

NH

HN NH2

D base

Modify Arg residue

H+

Add appropriately basic group

Cit NH

O NH2

D base

MS/MS

proton is mobile Induce the cleavage

of glycosidic bond

H+

D base

Cit

Arginine Removal

Arginine Modification

NH

O NH2

MALDI-MSn analysis

Glycoprotein

Trypsin digestion

Size exclusion chromatography

Carboxypeptidase B treatment

Tertiary amine labeling

Reversed phase HPLC

Peptidylarginine deiminase treatment

Arginine Removal Scheme

Arginine Modification Scheme

GluC digestion

R

R base

base

R

R base

Cit base

【M+H】+

: mannose : N-acetylglucosamine

: galactose :cross-ring cleavage of N-acetylglucosamine

1000 1500 2000 2500 3000 3500 4000 4500

X10

QDQC I YNTTYLNVQ

m/z

(b)

(a)

1000 1500 2000 2500 3000 3500 4000 4500 m/z

X10 X2

QDQC I YNTTYLNVQR

No derivatization

Tertiary amine labeling & arginine removal

MS/MS

TMT

1000 1500 2000 2500 3000 3500 4000 m/z

X20 X5 X20 (c)

N GT I SRYVGGQE

1000 1500 2000 2500 3000 3500 4000 m/z

-43D

a

X2 (d)

N GT I SCitYVGGQE TMT

No derivatization

Tertiary amine labeling & arginine modification

T5-2

T5-1 T2

T4-1, T4-2

T1-2* T1-1*

T3

T1-1

m/z

Retention Time / min