From: The Protein Protocols Handbook, Third EditionEdited by: J.M. Walker © Humana Press, a Part of Springer Science + Business Media, LLC 2009

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MS Analysis of Protein Glycosylation

Nobuaki Takemori, Naoka Komori, and Hiroyuki Matsumoto

1. Introduction

Post-translational modifications (PTM) are important molecular events because they generate functional diversities of proteins. A large number of proteins undergo multiple PTM in vivo and over 300 types of PTM have been identified to date (1). Among various PTM, phosphorylation and glycosylation have been intensely studied because of their physiological significance. Phosphorylation plays key regulatory roles in cellular signaling processes (2), and glycosylation mediates crucial cellular mechanisms such as protein folding and trafficking (3, 4). To confirm the presence of these modifications in vivo, the conventional analytical procedures require large quantities of samples and laborious bio-chemical steps. For this reason, small-scale analysis of PTM remains among the most challenging areas of biological science.

This chapter describes our recent proteomic methodology using the combina-tion of two-dimensional gel electrophoresis (2-DGE) and MALDI quadrupole ion trap time-of-flight mass spectrometer (MALDI-QIT-TOF MS), which enables a highly sensitive analysis of post-translational modifications (PTM). Two-DGE has been used extensively as a powerful tool to separate proteins from a limited amount of biological samples (5–8). We stained gels with Pro-Q™ Emerald/Diamond dyes immediately after 2-DGE and prior to CBB staining, and because this allows us to perform direct detection of glycoproteins/phosphoproteins on the same 2-D gels (9,10). Gel-separated glycoproteins are further subjected to the structural analysis using MALDI-QIT-TOF MS. MALDI-QIT-TOF MS is capable of structural characterization of various PTMs through highly sensi-tive fragmentation, or MS/MS, analysis (11,12). In MALDI-QIT-TOF MS, two mass analyzers (i.e., QIT and TOF) are connected to MALDI ion source, and the combination of MALDI and QIT renders a unique opportunity to perform multistage fragmentation (MSn) analysis with high-energy collision-induced

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dissociation (CID) (11,13). In this chapter, we will describe proteomic tech-niques that we use to analyze N-linked glycosylation. However, the protocol can be applied to other PTM analyses with some modifications.

2. Materials 1. MALDI-QIT-TOF MS (AXIMA QIT; Shimadzu Biotech, Manchester, UK) 2. MALDI-TOF MS 3. MALDI stainless sample target 4. Acetonitrile (HPLC grade) 5. Ammonium bicarbonate 6. Trifluoroacetic acid (TFA) 7. Milli-Q water (Millipore, Bedford, MA, USA) 8. Sequencing-grade trypsin (Promega Madison, WI, USA). 9. Stock solutions for in-gel digestion:

a. 50% (v/v) acetonitrile/100 mM ammonium bicarbonate (pH 8.9)b. 50% (v/v) acetonitrile/5% (v/v) TFAc. 0.2% (v/v) TFA solutiond. 10 mM ammonium bicarbonate (pH 8.9)

10. Recrystalized 2,5 dihydroxybenzoic acid (DHB) 11. DHB solution for MS analysis: 2% (w/v) DHB in 50% (v/v) acetonitrile/0.1%

(v/v) TFA (see Note 1)

3. Methods

Figure 1 shows a schematic diagram of our glycoprotein analysis. Protein samples are first separated using 2-DGE, and glycoprotein spots are detected using a glycoprotein staining reagent (Subheading 3.1). Glycoprotein spots are in-gel digested with trypsin (Subheading 3.2) (see Note 2), and identified by peptide mass fingerprinting analysis (Subheading 3.3). In order to characterize their glycan structures and modification sites, tryptic glycopeptides are further subjected to fragmentation analysis in MALDI-QIT-TOF MS (Subheading 3.4). Because human keratin contamination interferes with MS analysis, it is recom-mended that powder-free latex gloves be worn throughout the procedure.

3.1. Gel Electrophoresis and Glycoprotein Staining 1. Perform 2-DGE to resolve glycoproteins. We perform isoelectric focusing for the

1st dimension and SDS-PAGE for the 2nd dimension (see Note 3). Instead of 2-DGE, one-dimensional gel electrophoresis (e.g., SDS-PAGE) can be used for this protocol. However, the resolution of proteins on a one-dimensional gel is generally inadequate for the separation of complex protein mixtures.

2. Following 2-DGE, gels are stained with a glycoprotein staining reagent for detec-tion of glycoproteins (see Note 4) or followed by Coomassie Brilliant Blue (CBB) for visualization of all protein spots (see example in Fig. 2).

MS Analysis of Protein Glycosylation 1389

Gel Electrophoresis

CBB (Total Protein Staining)

Pro-Q Emerald (Glycoprotein Staining)

In-Gel Tryptic Digestion

m/z

Peptide Mass Fingerprinting by MALDI-TOF MS

Structural Characterizationby MALDI-QIT-TOF MS

SD

S-P

AG

E

IEF

Mannose (162 Da)

GlcNAc (203 Da)

Fucose (146 Da)

A B

C

D

Fig. 1. A schematic drawing of glycoprotein analysis using 2-DGE and MALDI-QIT-TOF MS. (A) Proteins are separated by 2-DGE based on their isoelectric focusing points and molecular weights in the first and second dimension, respectively. A glycosylated protein spots are detected by staining with Pro-Q Emerald dye. (B) After in-gel digestion of the glycoprotein spot with trypsin, the tryptic peptides are subjected to peptide mass finger-printing analysis. (C) Peptide mass fingerprinting analysis provides the mass profiles and sequence information of the tryptic peptides. (D) A glycopeptide thus detected is further fragmented by MALDI-QIT-TOF MS. Using the information from fragment ions, the struc-ture of a glycan moiety can be deduced.

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3.2. In-Gel Tryptic Digestion 1. After detection of protein spots positive for glycoprotein staining, correspond-

ing protein spots stained with CBB are excised by a razor blade and transferred into a 1.5 ml polypropylen tube. Excised gel pieces are shaken in 1 ml of 50% (v/v) acetonitrile/100 mM ammonium bicarbonate (pH 8.9) at room temperature to remove CBB.

2. Distained gel pieces are incubated with 1 ml of acetonitrile for 30 min to dehydrate the gels. After removing acetonitrile, the gel pieces are rehydrated with 2 μl of trypsin solution for 30 min at 4°C. Add 50 μl of 10 mM ammonium bicarbonate (pH 8.9) and incubate at 37°C for 5–10 hrs. During the incubation, trypsin continues

A Pro-Q Emerald

B CBB

Fig. 2. Differential staining of Drosophila proteins with a fluorescent glycosylation sensor dye, Pro-Q™ Emerald (A) and Coomassie blue (B). Drosophila eye proteins were separated on 2-D gel. Glycosylated protein spot (arrow) was identified by staining with Pro-Q Emerald dye. Our peptide mass fingerprinting analysis identified the spot as chaoptin.

MS Analysis of Protein Glycosylation 1391

to digest a protein in a gel piece and while some of the tryptic peptides come out of the gel piece.

3. Transfer the solution containing tryptic digests into a new 0.65 ml polypropylen tube. To extract the rest of the tryptic digests from the gel, vigorously shake the gel in 50 μl of 50% (v/v) acetonitrile/5% (v/v) TFA for 20 min. After a brief centri-fuge, collect and transfer the supernatant into the new tube to mix with the original digest solution.

4. Concentrate the collected peptide solution down to near dryness using vacuum centrifuge (see Note 5).

3.3. Peptide Mass Fingerprinting Analysis using MALDI-TOF MS 1. Each peptide sample is reconstructed with 5 μl of 0.2% (v/v) TFA solution. Load

the peptide solution (0.5 μl) onto a MALDI-TOF sample target plate with a fresh DHB solution (0.5 μl) and dry the mixture completely at room temperature.

2. Peptide samples on the MALDI sample target are subjected to peptide mass fingerprinting analysis using MALDI-TOF MS. After measuring the masses of the tryptic peptides by MALDI-TOF MS, the observed masses are submitted to MASCOT PMF search program (Matrix Science, London, UK) at http://matrixscience.com.

3. We routinely perform MASCOT search against the latest version of the National Center for Biotechnology Information (NCBI) nonredundant database using the following parameters; (a) unlimited protein molecular weight and pI ranges, (b) presence of protein modifications including acrylamide modification of cysteine, methionine oxidation, protein N-terminus acetylation, and pyroglutamic acid, and (c) mass tolerance of ± 0.25 Da. We consider the confirmation to be positive when a significant MOWSE score (p < 0.05) is generated.

4. After the protein identification by peptide mass fingerprinting analysis, the masses of the observed peptides are compared to that of the theoretical tryptic peptides. The peptides which contain glycans will be detected as the unassigned ion peaks because glycosylation leads to a predictable increase in the mass of a modified amino acid residue. When unassigned ion peaks are detected in MS spectrum, the masses of the unassigned peaks are subjected to ExPASy-GlycoMod Tool (http://au.expasy.org/tools/glycomod) to predict possible glycan structures and modification sites. To calculate the theoretical fragment ions resulting from CID fragmentation of the predicted peptide, we use MS-Product program (http://prospector.ucsf.edu/).

3.4. Structural Characterization of Protein Glycosylation Using MALDI-QIT-TOF MS

1. Load peptide solution (0.5 μl) onto a MALDI-QIT-TOF sample target with a fresh DHB solution (0.5 μl) and dry the mixture completely at room temperature. Each peptide sample is subjected to MALDI-QIT-TOF MS analysis.

2. In general, an ionized glycopeptide is fragmented without CID in MALDI-QIT-TOF MS, and as a result, a precursor glycopeptide together with some of its fragment ions will be detected in MS mode. Sugar components are readily

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2000 3000 4000

600 1000 1400 1800 2200

m/z

b16

P [1120-1136]

b16-H2O

y14y13y12y11

y11-H2Oy10

y10-H2O

y9

y9-H2O

y4

y5-H2O

y5

y7

y6-H2O

b10

0

100

y8-H2O

Rel

ativ

e In

tens

ity (

%)

Δ162 Δ486 (162 x 3)Δ203Δ203

2357.82154.91951.4

3978.5

Δ162 Δ162 Δ162 Δ162 Δ162 Δ162

P PP

B MS/MS 3978.5

C MS/MS 2154.9

A MS

1120 L TN I T F SGPQF TNLNE R 1136

y14 y4

b10 b16

P 7

2154.9

2520.3 3006.5 3168.5 3330.5 3492.6 3654.3 3816.7

Hexose (162 Da)HexNAc (203 Da)

3978.5

*

*

*3492.6

*3654.3 *3816.7

*3077.6

*2239.0

Fig. 3. Structural characterization of chaoptin glycopeptide using MALDI-QIT-TOF MS. The tryptic peptides of chaoptin (Drosophila glycoprotein) separated on 2-D gel (Fig. 2) were subjected to a series of CID fragmentation to deduce the glycan sequence and glyco-sylation site. (A) MS spectrum of the tryptic digests. *, peptide ions not assigned as tryptic peptides of chaoptin. (B) MS/MS spectrum of an unassigned ion peak at m/z 3978.5 observed in MS mode. CID fragmentation resulted in a series of fragment ions separated by 162, 203, or 486 (162 × 3) Da. (C) MS/MS spectrum of a fragment ion at m/z 2154.9. The y ion and b ion series derived from the peptide corresponded to the amino acid residues 1120 to 1136 of chaoptin. ≠, a putative N-linked glycosylation site. �, a peptide ion with a cross-ring cleav-age of HexNAc residue. � in dark gray, hexose residue; � in light gray, N-acetylhexosamine (HexNAc) residue. (With permission from ref. 11)

distinguishable from amino acid residues and other post-translationally modified residues based on their masses. As a result, we can easily distinguish glycopep-tides from other nonglycopeptides.

MS Analysis of Protein Glycosylation 1393

3. To determine the actual glycan structure, an ionized glycopeptide is further subjected to fragmentation analysis in MS/MS mode. As compared with peptide-bonds, gly-cosidic bonds are susceptible to CID fragmentation resulting in a sequential loss of sugar components from the terminal end of a glycan moiety (see example in Fig. 3).

4. Because some peptide-bonds seldom undergo total dissociation in a single run of MS/MS analysis, further fragmentation analysis by MSn analysis may be required to determine the amino acid sequence and modification site(s).

4. Notes

1. Prepare fresh DHB solution just before use. 2. Some glycoproteins are not susceptible to tryptic cleavage due to the steri-

cal hindrance of their glycans. Digestion with other proteases may improve the recovery of glycopeptides.

3. In our laboratory, isoelectric focusing gel electrophoresis is carried out using IPGphor (GE Healthcare/Amersham, Buckinghamshire, UK) with a cup loading strip holder. Immobiline Dry Strip (pH 3–10, 13 cm in length) is rehydrated for 15 hr at room temperature with 250 μl of lysis solution (8.5 M urea, 2% (w/v) CHAPS, 0.2% (w/v) Bio-Lyte 3/10 (BIO-RAD, Hercules, CA, USA), and 5% (v/v) β-mercaptoethanol). The same lysis solution (100 μl) is used for sample homogenization. After centrifugation at 15,000 × g for 10 min, the supernatant is loaded into a sample cup. Elec-trophoresis is performed at 500 V for 3 min, 4,000 V for 2 hr, and 8,000 V up to a total of 22,000 Vhr.

4. Several glycoprotein staining reagents are commercially available. We use Pro-Q™ Emerald glycoprotein gel stain (Molecular Probes, Eugene, OR, USA) because of its high sensitivity. Regarding the details of the Pro-Q stain-ing, refer to manufacture’s instructions.

5. Peptide samples can be stored at −20°C at least for several months.

References

1. Delta Mass, a database of protein post translational modifications (http://www.abrf.org/index.cfm/dm.home).

2. Graves, J. D. and Krebs, E. G. (1999) Protein phosphorylation and signal transduc-tion. Pharmacol. Ther. 82, 111–121,

3. Helenius, A. and Aebi, M. (2000) Intracellular functions of N-linked glycans. Science 291, 2364–2369.

4. Roseman, S. (2001) Reflections on glycobiology. J Biol Chem. 276, 41527–41542. 5. Matsumoto, H., O’Tousa, J. E., and Pak, W. L. (1982) Light-induced modification

of Drosophila retinal polypeptides in vivo. Science 217, 839–841. 6. Matsumoto, H. and Pak, W. L. (1984) Light-induced phosphorylation of retina-

specific polypeptides of Drosophila in vivo. Science 223, 184–186.

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7. Matsumoto, H., Kurien, B. T., Takagi, Y., Kahn, E. S., Kinumi, T., Komori, N., Yamada, T., Hayashi, F., Isono, K., Pak, W. L., Jackson, K. W., and Tobin, S. L. (1994) Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors. Neuron 12, 997–1010.

8. Komori, N., Takemori, N., Kim, H. K., Singh, A., Hwang, S. H., Foreman, R. D., Chung, K., Chung, J. M., and Matsumoto, H. (2007) Proteomics study of neuro-pathic and nonneuropathic dorsal root ganglia: Altered protein regulation follow-ing segmental spinal nerve ligation injury. Physiol. Genomics. 29, 215–230.

9. Hart, C., Schulenberg, B., Steinberg, T. H., Leung, W. Y., and Patton, W. F. (2003) Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 24, 588–598.

10. Steinberg, T. H., Agnew, B. J., Gee, K. R., Leung, W. Y., Goodman, T., Schulenberg, B., Hendrickson, J., Beechem, J. M., Haugland, R. P., and Patton, W. F. (2003) Glo-bal quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics 3, 1128–1144.

11. Takemori, N., Komori, N., and Matsumoto, H. (2006) Highly sensitive multistage mass spectrometry enables small-scale analysis of protein glycosylation from two-dimensional polyacrylamide gels. Electrophoresis 27, 1394–1406.

12. Takemori, N., Komori, N., Thompson, J. N. Jr, Yamamoto, M-T., and Matsumoto, H. (2007) Novel eye-specific calmodulin methylation characterized by protein map-ping in Drosophila melanogaster. Proteomics 7, 2651–2658.

13. Martin, R. L. and Brancia, F. L. (2003) Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer. Rapid Commun Mass Spectrom. 17, 1358–1365.