In-source supercharging and subcharging for glycopeptide ...€¦ · Pro instrument. Ionization...
1
For research use only. Not for use in Clinical diagnostic procedures. In-source supercharging and subcharging for glycopeptide analysis on the timsTOF pro platform Introduction Hans Wessels 1 , Kristina Marx 2 , Melissa Bärenfänger 1 , Merel Post 1 , Fokje Zijlstra 1 , Pierre-Olivier Schmit 2 , Alain van Gool 1 , Dirk Lefeber 1 1 Translational Metabolic Laboratory, Radboudumc, Nijmegen, The Netherlands 2 Bruker Daltonik GmbH, Bremen, Germany Recent introduction of parallel accumulation serial fragmentation (PASEF) on a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro) provides unique possibilities for comprehensive glycopeptide profiling in complex samples such as blood plasma. Poor ionization efficiency during electrospray ionization was solved previously by providing acetonitrile enriched nitrogen gas into the CaptiveSpray source via the nanoBooster. Here, we show first results using nanoBooster dopants for glycopeptide analysis on the timsTOF pro instrument. Conclusions • nanoBooster dopants are compatible with TIMS operation • Acetonitrile and primary alcohols enhance glycopeptide ionization efficiency • Acetonitrile dopant supercharges glycopeptide precursor ions and increases the number of charge states per precursor ion • Primary alcohols subcharge glycopeptide precursor ions and reduce charge state heterogeneity • PASEF enables comprehensive structural elucidation of glycopeptides timsTOF PASEF Methods Fig. 1: Effects of nanoBooster solvents compared to air for peptides and glycopeptides from human blood plasma. (A) : m/z versus 1/K0 precursor maps for peptides and glycopeptides with charge z=2 + up to z=6 + using air or ethanol and acetonitrile dopants. (B): Relative distributions of m/z, z, and 1/K0 values for all precursor ions for each nanoBooster condition. (C): Representative extracted ion currents (EIC) for each charge state of six randomly selected glycopeptide precursor ions. The fold change increase in signal intensity relative to air was calculated using the dominant charge state of each condition. Colors of the EIC traces correspond with charge states : blue: z=2 + , red: z=3 + , black: z=4 + , green: z=5 + Results Summary Use of acetonitrile and primary alcohols as nanoBooster dopant are compatible with TIMS and PASEF operation on the timsTOF Pro instrument. Ionization efficiency is significantly enhanced which enabled analysis of glycopeptides that were barely detectable under normal ESI source conditions (Fig1). PASEF using normal and elevated collision induced dissociation energies can be used to identify both the glycan- and peptide- moieties by GlycoQuest and MASCOT database searches as shown for selected IgG1 glycopeptides in figure 2. ASMS 2019 poster ThP 220 Blood plasma from healthy individuals was subjected to tryptic digestion and glycopeptides were enriched using Sepharose. Both the tryptic plasma digest and enriched glycopeptide fractions were measured by LC-IMS-MS/MS (Bruker Daltonics nanoElute and timsTOF Pro) using filtered air and dopant enriched nitrogen source gas using acetonitrile and ethanol as nanoBooster solvents. Data analysis was performed in DataAnalysis 5.0, ProteinScape 4.1, and in-house developed Perl scripts. Glycopeptide 2926.2 Da EtOH ACN Air 0 2 5 x10 Intens. 0 2 5 x10 Intens. 0 2 5 x10 Intens. 15.9 16.0 Time [min] Glycopeptide 6233.5 Da EtOH ACN Air 0 2 4 4 x10 Intens. 0 2 4 4 x10 Intens. 2 4 4 x10 Intens. 17.0 17.5 18.0 Time [min] 26.5x 22.2x 2+ 3+ 4+ 5+ 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 300 800 1300 1800 2300 2800 1/K0 m/z Peptides Glycopeptides_z2 Glycopeptides_z3 Glycopeptides_z4 Glycopeptides_z5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 300 800 1300 1800 2300 2800 1/K0 m/z Peptides Glycopeptides_z2 Glycopeptides_z3 Glycopeptides_z4 Glycopeptides_z5 Glycopeptides_z6 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 300 800 1300 1800 2300 2800 1/K0 m/z Peptides Glycopeptides_z2 Glycopeptides_z3 Glycopeptides_z4 Glycopeptides_z5 Glycopeptides_z6 0 10 20 30 40 50 60 2 3 4 5 6 Relative frequency (%) z Air EtOH ACN 0 2 4 6 8 10 12 14 16 18 300 500 700 900 1100 1300 1500 1700 1900 2100 2300 2500 2700 2900 Relative frequency (%) m/z Air EtOH ACN 0 5 10 15 20 25 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 Relative frequency (%) 1/K0 Air EtOH ACN A B Glycopeptide 3249.3 Da EtOH ACN Air 0 1 2 5 x10 Intens. 0 1 2 5 x10 Intens. 0 1 2 5 x10 Intens. 9.9 10.0 Time [min] 39.0x 26.0x 3.9x 3.1x C 0.0 0.5 1.0 1.5 2.0 5 x10 Intens. 0.0 0.5 1.0 1.5 2.0 5 x10 Intens. 0.5 1.0 1.5 2.0 5 x10 Intens. 30.8 31.0 31.2 Time [min] 4.0x 6.8x Glycopeptide 3681.5 Da 0.0 0.5 1.0 1.5 5 x10 Intens. 0.0 0.5 1.0 1.5 5 x10 Intens. 0.0 0.5 1.0 1.5 5 x10 Intens. 5.5 6.0 6.5 Time [min] 17.9x 20.8x Glycopeptide 3711.3 Da 0.00 0.25 0.50 0.75 5 x10 Intens. 0.00 0.25 0.50 0.75 5 x10 Intens. 0.00 0.25 0.50 0.75 5 x10 Intens. 23.0 23.2 Time [min] 2.0x 5.8x Glycopeptide 3639.4 Da Fig. 2: IgG glycopeptide identifications example.(A): Combined GlycoQuest and MASCOT search result of G0F with glycan and peptide moiety fragmentation. (B): Selection of the most abundant identified IgG1 glycopeptides is highlighted in the m/z versus 1/K0 precursor map. EtOH ACN Air 1.0 1.1 1.2 1.3 Mobility 1/K0 0 500 1000 1500 Int. 1100 1200 1300 1400 1500 1600 m/z B A This work was supported, in part, by the NWO ZonMw medium investment programme under project number 9118025.
Transcript of In-source supercharging and subcharging for glycopeptide ...€¦ · Pro instrument. Ionization...
For research use only. Not for use in Clinical diagnostic procedures.
In-source supercharging and subcharging for glycopeptide analysis on the timsTOF pro platform
Introduction
Hans Wessels1, Kristina Marx2, Melissa Bärenfänger1, Merel Post1, FokjeZijlstra1, Pierre-Olivier Schmit2, Alain van Gool1, Dirk Lefeber1
1Translational Metabolic Laboratory, Radboudumc, Nijmegen, The Netherlands2Bruker Daltonik GmbH, Bremen, Germany
Recent introduction of parallel accumulation serial fragmentation (PASEF) on a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro) provides unique possibilities for comprehensive glycopeptide profiling in complex samples such as blood plasma. Poor ionization efficiency during electrospray ionization was solved previously by providing acetonitrileenriched nitrogen gas into the CaptiveSpraysource via the nanoBooster. Here, we show first results using nanoBooster dopants for glycopeptide analysis on the timsTOF pro instrument.
Conclusions
• nanoBooster dopants are compatible with TIMS operation
• Acetonitrile and primary alcohols enhance glycopeptide ionization efficiency
• Acetonitrile dopant supercharges glycopeptide precursor ions and increases the number of charge states per precursor ion
• Primary alcohols subchargeglycopeptide precursor ions and reduce charge state heterogeneity
• PASEF enables comprehensive structural elucidation of glycopeptides
timsTOF PASEF
Methods
Fig. 1: Effects of nanoBooster solvents compared to air for peptides and glycopeptides from human blood plasma. (A): m/z versus 1/K0 precursor maps for peptides and glycopeptides with charge z=2+ up to z=6+ using air or ethanol and acetonitrile dopants. (B): Relative distributions of m/z, z, and 1/K0 values for all precursor ions for each nanoBooster condition. (C): Representative extracted ion currents (EIC) for each charge state of six randomly selected glycopeptide precursor ions. The fold change increase in signal intensity relative to air was calculated using the dominant charge state of each condition. Colors of the EIC traces correspond with charge states : blue: z=2+, red: z=3+, black: z=4+, green: z=5+
Results Summary
Use of acetonitrile and primary alcohols as nanoBooster dopant are compatible with TIMS and PASEF operation on the timsTOF Pro instrument. Ionization efficiency is significantly enhanced which enabled analysis of glycopeptides that were barely detectable under normal ESI source conditions (Fig1). PASEF using normal and elevated collision induced dissociation energies can be used to identify both the glycan- and peptide-moieties by GlycoQuest and MASCOT database searches as shown for selected IgG1 glycopeptides in figure 2.
ASMS 2019 poster ThP 220
Blood plasma from healthy individuals was subjected to tryptic digestion and glycopeptides were enriched using Sepharose. Both the tryptic plasma digest and enriched glycopeptide fractions were measured by LC-IMS-MS/MS (Bruker Daltonics nanoElute and timsTOF Pro) using filtered air and dopant enriched nitrogen source gas using acetonitrile and ethanol as nanoBooster solvents. Data analysis was performed in DataAnalysis 5.0, ProteinScape4.1, and in-house developed Perl scripts.
Glycopeptide 2926.2 DaEtOH
ACN
Air
0
2
5x10
Intens.
0
2
5x10
Intens.
0
2
5x10
Intens.
15.9 16.0 Time [min]
Glycopeptide 6233.5 DaEtOH
ACN
Air
0
2
4
4x10
Intens.
0
2
4
4x10
Intens.
2
4
4x10
Intens.
17.0 17.5 18.0 Time [min]
26.5x
22.2x
2+ 3+ 4+ 5+0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
300 800 1300 1800 2300 2800
1/K0
m/z
Peptides
Glycopeptides_z2
Glycopeptides_z3
Glycopeptides_z4
Glycopeptides_z5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
300 800 1300 1800 2300 2800
1/K0
m/z
Peptides
Glycopeptides_z2
Glycopeptides_z3
Glycopeptides_z4
Glycopeptides_z5
Glycopeptides_z6
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
300 800 1300 1800 2300 2800
1/K0
m/z
Peptides
Glycopeptides_z2
Glycopeptides_z3
Glycopeptides_z4
Glycopeptides_z5
Glycopeptides_z6
0
10
20
30
40
50
60
2 3 4 5 6
Relative
frequency(%)
z
Air
EtOH
ACN
0
2
4
6
8
10
12
14
16
18
300
500
700
900
1100
1300
1500
1700
1900
2100
2300
2500
2700
2900
Relative
frequency(%)
m/z
Air
EtOH
ACN
0
5
10
15
20
25
0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6
Relative
frequency(%)
1/K0
Air
EtOH
ACN
A B Glycopeptide 3249.3 Da EtOH
ACN
Air
0
1
2
5x10
Intens.
0
1
2
5x10
Intens.
0
1
2
5x10
Intens.
9.9 10.0 Time [min]
39.0x
26.0x
3.9x
3.1x
C
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
0.5
1.0
1.5
2.0
5x10
Intens.
30.8 31.0 31.2Time [min]
4.0x
6.8x
Glycopeptide 3681.5 Da
0.0
0.5
1.0
1.5
5x10
Intens.
0.0
0.5
1.0
1.55x10
Intens.
0.0
0.5
1.0
1.5
5x10
Intens.
5.5 6.0 6.5 Time [min]
17.9x
20.8x
Glycopeptide 3711.3 Da
0.00
0.25
0.50
0.75
5x10
Intens.
0.00
0.25
0.50
0.75
5x10
Intens.
0.00
0.25
0.50
0.75
5x10
Intens.
23.0 23.2 Time [min]
2.0x
5.8x
Glycopeptide 3639.4 Da
Fig. 2: IgG glycopeptide identifications example.(A): Combined GlycoQuest and MASCOT search result of G0F with glycan and peptide moiety fragmentation. (B): Selection of the most abundant identified IgG1 glycopeptides is highlighted in the m/zversus 1/K0 precursor map.
EtOH
ACN
Air
R.EEQYNSTYR.V
1.0
1.1
1.2
1.3
Mobility1/K0
0
500
1000
1500
Int.
1100 1200 1300 1400 1500 1600 m/z
B
A
This work was supported, in part, by the NWO ZonMw medium investment programme under project number 9118025.
