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.