Gold Nanoparticles as an Inorganic Matrix for MALDI-TOF Mass Spectrometry of Low

Molecular Weight Polymers

Justin R. Engle and S. Kim R. Williams

Laboratory for Advance Separations TechnologiesDepartment of Chemistry and Geochemistry

Colorado School of MinesGolden, Colorado

MALDI-TOF sample preparation

2,5-dihydroxybenzoic acid(DHB)

+

Deposition

OH

OHOH

O

MatrixAnalyte

Hn

Polystyrene (PS) 800

Mw 826

Mn 726

Polyethylene glycol (PEG) 700

HO

OHn Mw 700

Mn 645

Salt+

MALDI-TOF schematic

Applied Biosystems. Perseptive Biosystems Voyager DE-STR. Version 5.1 2001.

Perseptive Biosystem Voyager DE-STR with delay extraction option

Laser desorption/ionization process

•Matrix in higher concentration than analyte and salt

-Absorbs laser energy

-Transfers energy to analyte

•Multiple ionization processes at ablation site and in plume

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 00

2 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

P S - A g +

e n d g r o u p s : t - b u ty l a n d H +

P S - K +

e n d g r o u p s : t - b u ty l a n d O H -

n = 71 0 4m /z

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 00

7 0 0 0 0

DHB + PS 800 + AgTFAR

elat

ive

Inte

nsity

Uni

ts

m / z

P S - A g + ( n = 4 ,5 )e n d g r o u p s : t - b u t y l a n d H +

P S - A u + ( n = 3 ,4 )e n d g r o u p s : t - b u t y l a n d H +

P S - C u + ( n = 4 ,5 )e n d g r o u p s : t - b u t y l a n d H +

1 0 4m /z

Stainless Steel MALDI Plate

Gold MALDI Plate

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 00

6 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

n = 1 6

4 4 m /z

P E G - N a +

e n d g r o u p s : O H - a n d H +

P E G - L i+

e n d g r o u p s : O H - a n d H +

P E G - A g +

e n d g r o u p s : O H - a n d H +

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 00

3 0 0 0 0

m / z

P E G - N a +

e n d g r o u p s : O H - + H +

P E G - K +

e n d g r o u p s : O H - a n d H +

P E G - H +

e n d g r o u p s : O H - a n d H +

P E G - L i +

e n d g r o u p s : O H - a n d H +

P E G - A g +

e n d g r o u p s : O H - a n d H +

n = 1 4

4 4 m / z

Rel

ativ

e In

tens

ity U

nits

Stainless Steel MALDI Plate

Gold MALDI Plate

DHB + PEG 700 + NaTFA

Traditional approach results• Complicated mass spectra below 1000 Da• Multiple unexplainable mass peaks

Result from fragmentation of organic matrix

• Incomplete mass series for PSPS on

Stainless Steel

PS on Gold

PEG on Stainless

Steel

PEG on Gold

Calc Mw 945 ± 65 692 ± 34 671 ± 25 646 ± 35

Calc Mn 862 ± 53 647 ± 25 631 ± 18 603 ± 28

Averages and deviation taken from at least 5 different spectra

Objectives

• Replace organic matrix with gold nanoparticles (AuNP)

Reduce number of mass peaks below 1000 Da• Investigate difference in sample plates

Stainless steel and gold MALDI plates• Determine reproducibility• Calculate molecular weight averages

Gold nanoparticlesUsed in numerous fields– Absorbs light below 400 nm (UV range) – Fluorescence of light energy after excitation

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

190 290 390 490 590 690 790

Wavelength (nm)

Abs

orba

nce

(uni

ts)

AuNP (d=10nm)AuNP (d=5.3nm)

337 nm

Absorption band:spherical gold nanoparticles

Sample preparationAnalyte 5 mg/mLSalt 5 mg/mLAuNPs d=5.3 nm; d=10nm

from Corpuscular (Cold Spring, NY)

Mixed in 1:1:1 (v/v)PS-Ag+; PEG-Na+

0.5 μL spot volumes on either stainless steel or gold MALDI plates

Instrument settings:Acceleration voltage: 25,000 vDelay extraction time:100 ns Grid voltage: 96%Laser intensity: 1926 units

Software analysis:Data Explorer (PerSeptiveBiosystems, Farmington, MA)Polymerix (Sierra Analytics, Modesto, CA)

AuNP assisted laser desorption/ionization

• AuNPs absorb laser energy, emit light energy to analyte and/or salt

• Fewer particle interactions

Stainless steel and gold MALDI plates

0 250 500 750 10000

30000

Rel

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e In

tens

ity U

nits

m/z

0 250 500 750 10000

30000

Rel

ativ

e In

tens

ity U

nits

m/z

Cleaned using tetrahydrofuran, 6M nitric acid, DI water, and acetone (in order)

0 250 500 750 10000

35000

Rel

ativ

e In

tens

ity U

nits

m/z

Salts (Li+, Na+)

AuNPs on MALDI platesd=5.3nm on stainless steel plate

d =5.3nm on gold plate d =10nm on gold plate

0 250 500 750 10000

35000

Rel

ativ

e In

tens

ity U

nits

m/z

Salts (Li+, Na+, K+)

Au+

Au+2

d=10nm on stainless steel plate

0 250 500 750 10000

35000

Rel

ativ

e In

tens

ity U

nits

m/z

Salts (Li+, Na+)

Au+

0 250 500 750 10000

35000

Rel

ativ

e In

tens

ity U

nits

m/z

Salts (Li+, Na+)

AuNPs, PS 800, AgTFAon Stainless Steel

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

1 5 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

A g +

A u +

n = 8 1 0 4 m / z

P S - A g +

E n d g r o u p s : H + a n d t - b u t y lAuNP (d=10nm)

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

6 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

A g +

P S - A g +

E n d G r o u p s : H + a n d t - b u t y l

P S - N a +

E n d G r o u p s : H + a n d t - b u t y l

n = 8

n = 7

1 0 4 m / z

AuNP (d=5.3nm)

Hn

Polystyrene

Hn

Polystyrene

AuNPs, PS 800, AgTFAon gold plate

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

3 2 5 0 0

Rel

ativ

e In

tens

ity U

nits

m /z

P S -A g +

E n d g r o u p s : H + a n d t - b u ty l

n = 81 0 4 m /z

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

1 5 0 0 0

Rel

ativ

e In

tens

ity U

nits

m /z

P S - A g +

E n d g r o u p s : H + a n d t - b u ty l

A g +A g +

2

A g +3

n = 8 1 0 4 m /z

Hn

Polystyrene

Hn

Polystyrene

AuNP (d=10nm)

AuNP (d=5.3nm)

AuNPs, PEG 700, NaTFAon stainless steel

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 00

2 2 5 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

n = 1 44 4 m /z

P E G - N a +

E n d g r o u p s : H + a n d O H -

P E G - K +

E n d g r o u p s : H + a n d O H -

P E G - L i+

E n d g r o p u s : H + a n d O H -

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 00

5 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

P E G - N a +

E n d g r o u p s : H + a n d O H -

P E G - K +

E n d g r o u p s : H + a n d O H -

P E G - L i+

E n d g r o p u s : H + a n d O H -

n = 1 4

4 4 m / zH O

OHn

Polyethylene glycol

H OOHn

Polyethylene glycol

AuNP (d=10nm)

AuNP (d=5.3nm)

AuNPs, PEG 700, NaTFAon gold plate

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 00

2 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m /z

n = 1 4 4 4 m /z

P E G - N a +

E n d g r o u p s : H + a n d O H -

P E G - K +

E n d g r o u p s : H + a n d O H -

P E G - L i +

E n d g r o p u s : H + a n d O H -

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 00

2 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

n = 1 4 4 4 m / z

P E G - N a +

E n d g r o u p s : H + a n d O H -

P E G - K +

E n d g r o u p s : H + a n d O H -

P E G - L i +

E n d g r o p u s : H + a n d O H -

H OOHn

Polyethylene glycol

H OOHn

Polyethylene glycol

AuNP (d=10nm)

AuNP (d=5.3nm)

Reproducibility

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

6 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

6 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 00

6 0 0 0 0

Rel

ativ

e In

tens

ity U

nits

m / z

Calculated molecular weightsPS

Mw = 826Mn = 726

AuNP(d=5nm)Stainless

Steel

AuNP(d=10nm)Stainless

Steel

AuNP(d=5nm)

Gold plate

AuNP(d=10nm)Gold plate

Mw 816 ± 6 835 ± 27 826 ± 8 791 ± 7

683 ± 3Mn 717 ± 2 725 ± 16 725 ± 6

PEGMw = 700Mn = 645

AuNP(d=5nm)Stainless

Steel

AuNP(d=10nm)Stainless

Steel

AuNP(d=5nm)

Gold plate

AuNP(d=10nm)Gold plate

Mw 608 ± 6 688 ± 46 684 ± 18 654 ± 28

609 ± 34 Mn 567 ± 6 638 ± 36 633 ± 16

Averages and deviation taken from at least 5 different spectra

Conclusions

• AuNPs promoted desorption/ionization of low molecular weight polymers

• Limited background interference• Reproducible results• “Best” results obtained with specific mixtures

– Polystyrene• AuNP (d=10nm) on stainless steel plate• AuNP (d=5.3nm) on gold plate

– Polyethylene Glycol• AuNP (d=5.3nm) on gold plate

Acknowledgments• NSF-CHE-0515521• Williams Group

– Dr. Dean Lee – Dr. Ilyong Park– Dr. Claudia Lohmann– J. Ray Runyon – Adam Goering