intact glycopeptide analysis by trapped ion mobility tandem mass … · 2020. 9. 2. · (pasef) on...

For research use only. Not for use in diagnostic procedures.

IntroductionRecent 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 glycopeptideprofiling in complex samples such as blood plasma. Poor ionization efficiency during electrospray ionization was solved previously by providing acetonitrile enriched 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 subcharge glycopeptide precursor ions and reduce charge state heterogeneity

• PASEF enables comprehensive structural elucidation of glycopeptides

MethodsBlood plasma from healthy individuals was subjected to tryptic digestion and glyco-peptides were enriched using Sepharose. Both the tryptic plasma digest and enriched glycopeptide fractions were measured by LC-IMS-MS/MS (Bruker Daltonics nanoEluteand 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.

Intact glycopeptide analysis by trapped ion mobility tandem mass spectrometry

HUPO2019 poster 65265Hans Wessels1, Kristina Marx2, Melissa Bärenfänger1, Merel Post1, Fokje Zijlstra1, Pierre-Olivier Schmit2, Alain van Gool1, Jaran Jainhuknan3, Dirk Lefeber1

1Translational Metabolic Laboratory, Radboudumc, Nijmegen, The Netherlands2Bruker Daltonik GmbH, Bremen, Germany3Bruker Thailand

timsTOF PASEF

BA

EtOH

ACN

Air

0

2

5x10Intens.

0

2

5x10Intens.

0

2

5x10Intens.

15.9 16.0 Time [min]

EtOH

ACN

Air

02

44x10

Intens.

02

44x10

Intens.

2

44x10

Intens.

17.0 17.5 18.0Time [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

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300 800 1300 1800 2300 2800

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m/z

Peptides

Glycopeptides_z2

Glycopeptides_z3

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Glycopeptides_z5

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0,9

1

1,1

1,2

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300 800 1300 1800 2300 2800

1/K0

m/z

Peptides

Glycopeptides_z2

Glycopeptides_z3

Glycopeptides_z4

Glycopeptides_z5

Glycopeptides_z6

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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

Rela

tive

freq

uenc

y(%

)

z

Air

EtOH

ACN

0

2

4

6

8

10

12

14

16

18

300

400

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Rela

tive

freq

uenc

y(%

)

m/z

Air

EtOH

ACN

0

5

10

15

20

25

0,6

0,65 0,

7

0,75 0,

8

0,85 0,

9

0,95 1

1,05 1,

1

1,15 1,

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1,25 1,

3

1,35 1,

4

1,45 1,

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1,55 1,

6

Rela

tive

freq

uenc

y(%

)

1/K0

Air

EtOH

ACN

A BEtOH

ACN

Air

0

1

25x10

Intens.

0

1

25x10

Intens.

0

1

25x10

Intens.

9.9 10.0 Time [min]

39.0x

26.0x

3.9x

3.1x

C

0.00.51.01.52.05x10

Intens.

0.00.51.01.52.05x10

Intens.

0.51.01.52.05x10

Intens.

30.8 31.0 31.2Time [min]

4.0x

6.8x

0.00.5

1.0

1.55x10

Intens.

0.00.5

1.0

1.55x10

Intens.

0.0

0.5

1.0

1.55x10

Intens.

5.5 6.0 6.5 Time [min]

17.9x

20.8x

0.000.250.500.75

5x10Intens.

0.000.250.500.75

5x10Intens.

0.000.250.500.75

5x10Intens.

23.0 23.2 Time [min]

2.0x

5.8x

EtOH

ACN

Air

Glycopeptide 2926.2 Da

Glycopeptide 6233.5 Da

Glycopeptide 3249.3 Da Glycopeptide 3681.5 Da

Glycopeptide 3711.3 Da

Glycopeptide 3639.4 Da

Results

SummaryUse 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.

HUPO2019

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 glycopeptideprecursor 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+

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.

This work was supported, in part, by the NWO ZonMw medium investment programme under project number 9118025.