ASMS 2010 poster
1
50 100 Relative Abundance (%) 50 100 Relative Abundance (%) 500 700 900 1100 1300 1500 1700 1900 (m/z) 1054.5 1157.7 1216.6 1319.7 1460.8 1232.6 1378.8 Bead Preparation and Protein Deglycosylation Purification and Deglycosylation of YCT721 Spent Culture Medium from Kluyveromyces lactis containing protein YCT721 (500 mL; A600= 21.3 OD/ mL) was put over a 5 mL HiTrap ProteinA HP column. The sample was washed with 10 column volumes with binding buffer (1X PBS, 1 mM EDTA, pH 7.4) and YCT721 was eluted with 8 column volumes using 0.1 M Citric acid (pH 2.85) with 2 mL fractions, each containing 400 μL 1.5 M Triethanolamine (pH 8.8). The protein was then concentrated and buffer exchanged into 10 mM Tris-HCl (pH 7.0) using Vivaspin 10K MWCO. The deglycosylation of YCT721 with Endo H-bound magnetic beads was conducted as described. Analysis by Mass Spectrometry Supernatant from the deglycosylation reaction (8 μL) was loaded onto a PLRP-S column, washed and then eluted by a step-gradient for online analysis of proteins (6210 TOF LC/MS, Agilent Technologies). To analyze the sugars, supernatants were spotted at a 1:1 ratio with matrix (Super-DHB) and then analyzed by MALDI MS (MALDI micro MX, Waters Corporation). C. Madinger , E. Guthrie, M. Ganatra, C. Taron, J. Benner New England Biolabs, Ipswich, MA 01938, USA Rapid deglycosylation of proteins for carbohydrate and protein characterization by mass spectrometry using Endo H-bound magnetic beads www.neb.com WP 296 Conclusions Overview Results Introduction Protein glycosylation is a prevalent post-translational modification. In fact, in mammalian systems 50-90% of all proteins are glycosylated. Glycosylation is involved in many diverse biological systems and can be required for proper protein folding and to protect proteins against proteolysis and thermal denaturation. Additionally, glycans on proteins are involved in very specific responses: they participate in the immune response and change with the condition of the cell/tissue (i.e., glycans on tumor cells are different from glycans on normal cells). The methodologies for protein deglycosylation are often time consuming and require detergents that obscure both the protein and carbohydrate. Here we descr be a rapid, efficient and undemanding method for analyses of both glycosylation and the deglycosylated, scarred protein. Methods Intact ovalbumin was analyzed online by ESI-MS before and after its incubation with Endo H-bound Magnetic Beads. Before treatment (Figure 1A), the sample is complex and contains several different species with masses of 44167.4, 44329.5, 44573.5 and 44776.9 Da in the approximate ratio of 19:38:31:12, respectively. Deglycosylated protein (43153.2 Da) is not detected initially, however it is detected after a 10 minute incubation with Endo H (Figures B1 and C1). The sample remains complex even with overnight incubation (Figures B2 and C2) and the percent of deglycosylated ovalbumin is approximately equal when deglycosylation was conducted with both Endo H-bound Magnetic Beads and Endo H in-solution (28% versus 21%, respectively). After incubation with Endo H-bound Magnetic Beads, the ovalbumin sample was then analyed by MALDI-TOF to detect the enzymatically-removed sugars. A complex spectrum was detected, with major ions at m/z 1054.4 1+ and 1216.4 1+ corresponding to Man 5 GlcNAc and Man 6 GlcNAc, respectively (Figure D1). Sugars with a single GlcNAc (i.e., Man 5 GlcNAc and Man 6 GlcNAc ) are anticipated products from the reaction of an N-glycosylated protein with Endo H since Endo H cleaves within the chitobiose core, leaving a GlcNAc scar on the protein. In contrast, most l kely due to signal suppression or ion competition, no sugars were detected from the ovalbumin sample after incubation with Endo H in-solution (Figure D2). In contrast, RNase B (NEB #P78117S) was also digested with Endo H (both bound to beads and in-solution) and the protein was completely deglycosylated with both conditions after only 30 min (data not shown). However, similar results were observed with the sugar analysis: no sugars were detected when RNase B digested with Endo H in-solution was analyzed. - Endo H is a good glycosidase to use for glycoproteomics. The enzyme is active both at low pH and with buffers that won’t suppress MS signal. Additionally, the reaction of Endo H leaves a GlcNAc scar on the protein thereby making glycosylation sites identifiable by MS/MS analyses. - The activity of Endo H is affected by the protein substrate: the deglycosylation of ova bumin was incomplete even after an overnight incubation whereas RNase B was completely deglycosylated after 30 min. - With the Endo H-bound Magnetic Beads both intact protein and deglycosylated sugar analyses can be conducted from the same sample without any additional sample preparation. In contrast, Endo H in-solution poses a problem because the enzyme can co-elute with the deglycosylated substrate when using a “step- gradient”. Additionally, to identify sugars resulting from deglycosylation with Endo H in-solution, further clean up using Carbon Graphite Zip Tips is required (data not shown). 1702.8 1703.8 1704.7 1705.7 Relative Intensity (%) 100 50 m/z 1650 1700 1750 1800 1850 1900 Deconvoluted Mass (Da) 50000 52000 54000 56000 58000 60000 62000 64000 56264 5 57998 8 59589.1 61346.8 62686.1 64441 0 Counts (x10 4 ) Counts (x10 4 ) 0.2 0.4 0.6 0.8 1 1 2 3 5 4 59751.2 59913.2 60002.3 Results To test the functionality of the Endo H-bound Magnetic Beads on experimental samples, YCT721, a glycosylated protein from Kluyveromyces lactis, was analyzed. The intact, glycosylated mass of YCT721 was 59,589.02 Da (Figure 2A) and this mass shifts to 56,264.5 Da (Figure 2B) after incubation overnight with Endo H-bound Magnetic Beads. Endo H is known to cleave within the chitobiose core of N-linked glycoproteins leaving a GlcNAc scar on the deglycosylated protein (Figure 2A, insert). In support, a sugar with a single GlcNAc (sodiated Man 9 GlcNAc, Figure 2A insert, m/z 1702.8 1+ ) was detected in the glycan analysis of YCT721 after overnight incubation with Endo H-bound Magnetic Beads. Examination of the intact protein mass before and after deglycosylation combined with the detection of Man 9 GlcNAc suggests that YCT721 has two Man 9 GlcNAc 2 modifications (Figure 2B, insert). Interestingly, however, YCT721 only has one glycosylation site and the mass of the intact, deglycosylated protein (Figure 2B) is significantly greater than predicted by the amino acid sequence (data not shown). Because of these MS data, YCT721 was identified as a self-associating dimer. 59589.1 – 56230.5 = 3358.5 56264.5 -2(17) 3358.5 – 1678.7 = 1679.8 1702.8 -23 Figure 2. Spectra from the MS analysis of YCT721 before (A) and after (B) deglycosylation with Endo H-bound magnetic beads. Spectrum from the glycan analysis of YCT721 is insert to (A), the mechanism for the removal of Man 9 GlcNAc from YCT721 via Endo H is insert to (A), and the breakdown demonstrating that two Man 9 GlcNAc 2 are bound to YCT721 is insert to (B). A. B. Acknowledgments The authors are grateful to Don Comb and New England Biolabs for their support of this work. And we would also like to thank Paula Magnelli for her assistance and technical expertise. 43152.7 43152.9 44329.5 44573.4 Deconvoluted Mass (Da) 40,000 42,000 44,000 46,000 48,000 50 100 44167.5 44573.4 44776 6 44329.4 44167.5 44777.4 Relative Abundance (%) Relative Abundance (%) 50 100 42,000 44,000 46,000 48,000 40,000 Deconvoluted Mass (Da) 43153.2 44574 3 44168.0 44329.2 44776 3 Relative Abundance (%) 50 100 43156 6 44573.3 44168.4 44329.3 44777.6 Relative Abundance (%) 50 100 48,000 46,000 44,000 42,000 40,000 Deconvoluted Mass (Da) Relative Abundance (%) 50 100 44329.5 44167.4 445573 5 44776 9 Endo H-bound magnetic beads Endo H in solution 10 min 16 hrs 10 min 16 hrs 16 hrs 16 hrs Endo H-bound magnetic beads Endo H in-solution Figure 1. Spectra from the MS analysis of ovalbumin before (A) and after deglycosylation with Endo H both in- solution (B) and bound to magnetic beads (C) at 10 min and 16 hrs (B1 & B2 and C1 & C2, respectively). Spectra from the glycan analysis of ovalbumin after overnight digestion with Endo H bound to magnetic beads (D1) and in solution (D2) is also shown. A. B1. B2. C1. C2. D1. D2. - Glycosylation is a prevalent post-translational modifications that has significant biological relevance - Deglycosylation of proteins using glycolytic enzymes are often time-consuming and require post-reaction sample clean up for successful product analysis by MS - By immobilizing Endo H onto magnetic beads, the glycolytic enzyme can be easily removed after the reaction thus enabling MS analyses of both the deglycosylated protein and the removed sugar from the same sample.
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Transcript of ASMS 2010 poster
50
100
Re
lative
Ab
un
da
nce
(%
)
50
100
Re
lative
Ab
un
da
nce
(%
)
500 700 900 1100 1300 1500 1700 1900
(m/z)
1054.5
1157.7
1216.6
1319.7 1460.8
1232.6 1378.8
Bead Preparation and Protein Deglycosylation
Purification and Deglycosylation of YCT721 Spent Culture Medium from Kluyveromyces lactis containing protein YCT721 (500 mL; A600= 21.3 OD/ mL) was put over a 5 mL HiTrap ProteinA HP column. The sample was washed with 10 column volumes with binding buffer (1X PBS, 1 mM EDTA, pH 7.4) and YCT721 was eluted with 8 column volumes using 0.1 M Citric acid (pH 2.85) with 2 mL fractions, each containing 400 µL 1.5 M Triethanolamine (pH 8.8). The protein was then concentrated and buffer exchanged into 10 mM Tris-HCl (pH 7.0) using Vivaspin 10K MWCO. The deglycosylation of YCT721 with Endo H-bound magnetic beads was conducted as described.
Analysis by Mass Spectrometry Supernatant from the deglycosylation reaction (8 µL) was loaded onto a PLRP-S column, washed and then eluted by a step-gradient for online analysis of proteins (6210 TOF LC/MS, Agilent Technologies). To analyze the sugars, supernatants were spotted at a 1:1 ratio with matrix (Super-DHB) and then analyzed by MALDI MS (MALDI micro MX, Waters Corporation).
C. Madinger, E. Guthrie, M. Ganatra, C. Taron, J. Benner
New England Biolabs, Ipswich, MA 01938, USA
Rapid deglycosylation of proteins for carbohydrate and protein characterization by mass spectrometry using Endo H-bound magnetic beads
www.neb.com
WP 296
Conclusions
Overview
Results
Introduction
Protein glycosylation is a prevalent post-translational modification. In fact, in mammalian systems 50-90% of all proteins are glycosylated. Glycosylation is involved in many diverse biological systems and can be required for proper protein folding and to protect proteins against proteolysis and thermal denaturation. Additionally, glycans on proteins are involved in very specific responses: they participate in the immune response and change with the condition of the cell/tissue (i.e., glycans on tumor cells are different from glycans on normal cells). The methodologies for protein deglycosylation are often time consuming and require detergents that obscure both the protein and carbohydrate. Here we descr be a rapid, efficient and undemanding method for analyses of both glycosylation and the deglycosylated, scarred protein.
Methods
Intact ovalbumin was analyzed online by ESI-MS before and after its incubation with Endo H-bound Magnetic Beads. Before treatment (Figure 1A), the sample is complex and contains several different species with masses of 44167.4, 44329.5, 44573.5 and 44776.9 Da in the approximate ratio of 19:38:31:12, respectively. Deglycosylated protein (43153.2 Da) is not detected initially, however it is detected after a 10 minute incubation with Endo H (Figures B1 and C1). The sample remains complex even with overnight incubation (Figures B2 and C2) and the percent of deglycosylated ovalbumin is approximately equal when deglycosylation was conducted with both Endo H-bound Magnetic Beads and Endo H in-solution (28% versus 21%, respectively).
After incubation with Endo H-bound Magnetic Beads, the ovalbumin sample was then analyed by MALDI-TOF to detect the enzymatically-removed sugars. A complex spectrum was detected, with major ions at m/z 1054.41+ and 1216.41+ corresponding to Man5GlcNAc and Man6GlcNAc, respectively (Figure D1). Sugars with a single GlcNAc (i.e., Man5GlcNAc and Man6GlcNAc ) are anticipated products from the reaction of an N-glycosylated protein with Endo H since Endo H cleaves within the chitobiose core, leaving a GlcNAc scar on the protein. In contrast, most l kely due to signal suppression or ion competition, no sugars were detected from the ovalbumin sample after incubation with Endo H in-solution (Figure D2).
In contrast, RNase B (NEB #P78117S) was also digested with Endo H (both bound to beads and in-solution) and the protein was completely deglycosylated with both conditions after only 30 min (data not shown). However, similar results were observed with the sugar analysis: no sugars were detected when RNase B digested with Endo H in-solution was analyzed.
- Endo H is a good glycosidase to use for glycoproteomics. The enzyme is active both at low pH and with buffers that won’t suppress MS signal. Additionally, the reaction of Endo H leaves a GlcNAc scar on the protein thereby making glycosylation sites identifiable by MS/MS analyses.
- The activity of Endo H is affected by the protein substrate: the deglycosylation of ova bumin was incomplete even after an overnight incubation whereas RNase B was completely deglycosylated after 30 min.
- With the Endo H-bound Magnetic Beads both intact protein and deglycosylated sugar analyses can be conducted from the same sample without any additional sample preparation. In contrast, Endo H in-solution poses a problem because the enzyme can co-elute with the deglycosylated substrate when using a “step-gradient”. Additionally, to identify sugars resulting from deglycosylation with Endo H in-solution, further clean up using Carbon Graphite Zip Tips is required (data not shown).
1702.8
1703.8
1704.7
1705.7
Re
lative
In
ten
sity (
%)
100
50
m/z
1650 1700 1750 1800 1850 1900
Deconvoluted Mass (Da)
50000 52000 54000 56000 58000 60000 62000 64000
56264 5
57998 8
59589.1
61346.862686.1
64441 0
Counts
(x10
4)
Counts
(x10
4)
0.2
0.4
0.6
0.8
1
1
2
3
5
4
59751.2
59913.2
60002.3
Results To test the functionality of the Endo H-bound Magnetic Beads on experimental samples, YCT721, a
glycosylated protein from Kluyveromyces lactis, was analyzed. The intact, glycosylated mass of YCT721 was 59,589.02 Da (Figure 2A) and this mass shifts to 56,264.5 Da (Figure 2B) after incubation overnight with Endo H-bound Magnetic Beads. Endo H is known to cleave within the chitobiose core of N-linked glycoproteins leaving a GlcNAc scar on the deglycosylated protein (Figure 2A, insert). In support, a sugar with a single GlcNAc (sodiated Man9GlcNAc, Figure 2A insert, m/z 1702.81+) was detected in the glycan analysis of YCT721 after overnight incubation with Endo H-bound Magnetic Beads. Examination of the intact protein mass before and after deglycosylation combined with the detection of Man9GlcNAc suggests that YCT721 has two Man9GlcNAc2 modifications (Figure 2B, insert). Interestingly, however, YCT721 only has one glycosylation site and the mass of the intact, deglycosylated protein (Figure 2B) is significantly greater than predicted by the amino acid sequence (data not shown). Because of these MS data, YCT721 was identified as a self-associating dimer.
59589.1 – 56230.5 = 3358.5
56264.5 -2(17)
3358.5 – 1678.7 = 1679.8
1702.8
-23
Figure 2. Spectra from the MS analysis of YCT721 before (A) and after (B) deglycosylation with Endo H-bound magnetic beads. Spectrum from the glycan analysis of YCT721 is insert to (A), the mechanism for the removal of Man9GlcNAc from YCT721 via Endo H is insert to (A), and the breakdown demonstrating that two Man9GlcNAc2 are bound to YCT721 is insert to (B).
A.
B.
Acknowledgments
The authors are grateful to Don Comb and New England Biolabs for their support of this work. And we would also like to thank Paula Magnelli for her assistance and technical expertise.
43152.7
43152.9
44329.5
44573.4
Deconvoluted Mass (Da)
40,000 42,000 44,000 46,000 48,000
50
100
44167.5
44573.4
44776 6
44329.4
44167.5 44777.4
Rela
tive
Abundance (
%)
Rela
tive
Abundance (
%)
50
100
42,000 44,000 46,000 48,00040,000
Deconvoluted Mass (Da)
43153.2 44574 3
44168.0
44329.2
44776 3
Rela
tive
Abundance (
%)
50
100
43156 6
44573.3
44168.4
44329.3
44777.6
Rela
tive
Abundance (
%)
50
100
48,00046,00044,00042,00040,000
Deconvoluted Mass (Da)
Rela
tive
Abundance (
%)
50
10044329.5
44167.4
445573 5
44776 9
Endo H-bound
magnetic beads
Endo H
in so
lution
10 min
16 hrs
10 min
16 hrs
16 hrs
16 hrs
Endo H-bound magnetic beads
Endo H in-solution
Figure 1. Spectra from the MS analysis of ovalbumin before (A) and after deglycosylation with Endo H both in-solution (B) and bound to magnetic beads (C) at 10 min and 16 hrs (B1 & B2 and C1 & C2, respectively). Spectra from the glycan analysis of ovalbumin after overnight digestion with Endo H bound to magnetic beads (D1) and in solution (D2) is also shown.
A.
B1.
B2.
C1.
C2.
D1.
D2.
- Glycosylation is a prevalent post-translational modifications that has significant biological relevance
- Deglycosylation of proteins using glycolytic enzymes are often time-consuming and require post-reaction sample clean up for successful product analysis by MS
- By immobilizing Endo H onto magnetic beads, the glycolytic enzyme can be easily removed after the reaction thus enabling MS analyses of both the deglycosylated protein and the removed sugar from the same sample.
