Analysis of Biomolecules through Nanomaterials Based Mass Spectrometry

Huan-Tsung ChangDepartment of ChemistryDepartment of Chemistry

National Taiwan UniversityNational Taiwan Universitychanght@ntu.edu.tw

07, 15, 2015

Optical

High absorption coefficientConcentration effectMore than thousand (at least 10X) molecules per NPsEasy sample preparationMinimum problems associated with sweet spots

Nanomaterials are used as the matrix in MS.

: GSH, Cys, and HCys; : Arg

(B)

Mixtures

(A)

AuAuAuAuAuAu hν

m/z

AuAu

AuAuAuAu

hν

m/z

Gold nanoparticles as a matrix for the quantitationof thiol compounds

Sig

nal

In

ten

sity

(a.

u.)

Concentration (M)

0 10 20 30 40 50 60

0 20 40 60 8010 30 50 70 90

0 60 8020 40 100 120

0

20

40

60

80

0

80

120

140

40

0

80

120

160

40

R2 = 0.997

R2 = 0.996

R2 = 0.995

(A)

(B)

(C)

Calibration Curves(A) [GSH + Na]+: 330.07

(B) [Cys + H]+: 143.98

(C) [HCys + H]+: 173.98

Linear Range

GSH : 2.5 － 50 M Hcys : 2.5 － 100 M Cys : 5.0 － 75 M

Limit of Detection GSH :1.0 M Hcys : 2.0 M Cys : 1.3 M

0_O7_11SRef

200

400

600

800

1000

Inte

ns. [

a.u.

]

0.1uM GSH50506

200

400

600

800

1000

Inte

ns. [

a.u.

]

0_P10_15SRef

0

200

400

600

800

Inte

ns. [

a.u.

]

325.0 327.5 330.0 332.5 335.0 337.5 340.0 342.5 345.0 347.5

m/z

(A)

(B)

346.03

335 340 345 350

m/z

Sig

nal

In

ten

sity

(a.

u.)

325

1

1

0

330.07

330

(C)10

0

330.07341.26

341.26

Concentration effect

1.0 M GSH

0.1 M GSH

0.1 M GSH

A-B: (NR)Au NPs (3) as matrices without concentrationC: 0.1 (NR)Au NPs -- concentration factor of 50

LOD: nM level

20 μL

1 mL

AuAu

AuAu

AuAu

ATP AuNP

(NH4)3citrate AuAu

AuAu

AuAu

AuAu

hν

m/z

AuAuAuAu

AuAu

Detection of ATP using Apt-AuNPs

: ATP-aptamer : ATP

Determination of ATP and GSH in Red Blood Cells

Apt-AuNP + AuNP

Apt-AuNP + AuNP

DHB

DHB

0.94 (± 0.06) mM (n = 3)

1.9 (± 0.3) mM (n = 3)

ATP

GSH

4-MBA4-Mercaptobenzoic acid (4-MBA)

Improved quantitation of CAP using 4-MBA as an internal standard

CAP

A drug for treatment of hypertensionCAP:

SALDI Mass spectrum of CAP, MBA and 14-nm Au NPs (7.5 nM) prepared in 0.1 mM ammonium citrate (pH 6.00).

[MBA － H] －

[CAP － H] －

Linearity: 2.5–25 μM

LOD: 1 uM

[CAP]

(M)

  With MBA-Au NPs (n = 3)   Without MBA-Au NPs (n = 3)

 

Found ± SD

(M)

Recovery

(%)

RSD

(%) 

Found ± SD

(uM)

Recovery

(%)

RSD

(%)

50   47.1 ± 1.50 94.2 8.3   39.5 ± 2.63 79 22

200   192.0 ± 3.20 96.0 5.7   115.0 ± 10.00 58 18

Energy-dispersive X-ray (EDX)UV-Vis

5 mM BSA

Absorbed light over UV-Vis range

Existence of HgTe

SA as matrixDominated peak:

[M + H]+

Laser shots : 300; laser pulse energy :62.5 μJ (power density :2 × 109 W/cm2).

Detection of Proteins through MS using HgTe

LOD: 14 nM

Mass limit : IgG (Mass limit : IgG (m/zm/z ~ 150 kDa) ~ 150 kDa)

Quasimolecular ions of two identical light chains of IgG

150 kDa ions detected

IgG (5 M) 12

HgTe has a low melting temperature and low thermal conductivity.Generation of higher temperature can be achieved for efficient desorption/ionization of the analytes.

Detection of protein-protein complexesDetection of protein-protein complexes

w/o Brij 76 w/ 1% Brij 76

Enhance 6-fold

w/ 0.1% Brij 76

w/ 0.1% Brij 76

w/o Brij 76

IgG + protein G

IgG

protein G

IgG: 5 uM (2.5 pmol)

protein G: 2 uM (1 pmol)LOD:1-antitrypsin: 3 uM (1.5 pmol)

Trypsin: 0.5 uM (0.25 pmol)

5 uM a1-antitrypsin

5 uM 1-antitrypsin + 1.7 uM trypsin

1.7 uM trypsin

LODs (from standard solutions)

Fructose: 15 μM Maltose: 10 μM

Longan honey

Fructose

Maltose

Detection of carbohydrates in vrious honey samples

(a) longan honey 1, (b) litchi honey, (c) osmanthus honey, (d) longan honey 2, (e) pomelo honey

pomelo honey

m/z 650–2700 Da

Oligosaccharides?

0

2000

4000

6000

In

ten

s. [a.u

.]

970 980 990 1000 1010 1020m/z

970 1030

8000995.8

0

1000

2000

3000

In

ten

s. [a.u

.]

900 950 1000 1050 1100 1150 1200 1250 1300 1350m/z

900 1400

4000

996.1 1158.01320.3

m/z0

0

Sig

nal in

ten

sity

(a.

u.)

[-CD + Na]+

[-CD + Na]+ [-CD + Na]+ [-CD + Na]+

0.5 mM ammonium citrate, pH 7.0 + 0.1 mM NaCl, 1X HgTe

0

500

1000

1500

2000

2500

3000In

ten

s. [a.u

.]

340 350 360 370 380 390m/z

[Sucrose + Na]+365.3

400

3000

0340

SALDI mass spectra of sucrose, SALDI mass spectra of sucrose, -CD, -CD, -CD, and -CD, and --CD CD

17

0

200

400

600

800

1000

In

ten

s. [a.u

.]

4000 4500 5000 5500 6000 6500 7000 7500m/z

5893.95406.6

6377.44919.7

4432.36861.1 m/z

0

50

100

150

200

Inte

ns.

[a.u

.]

4000 4500 5000 5500 6000 6500 7000 7500m/z

100

04000 8000

Sig

nal i

nten

sity

(a.

u.)

4000 5000 6000 7000 8000

0.5 mM ammonium citrate, pH 7.0

0

7347.5

1500

100 nM PL-6k

Laser : 75 J

Matrix : 1x HgTe

0

200

400

600

800

1000

In

ten

s. [a.u

.]

6000 7000 8000 9000 10000 11000m/z

7838 83218803

9285

9767

7356

6871

10248

10727

6387

11211

0

10

20

30

Inte

ns.

[a.u

.]

6000 7000 8000 9000 10000 11000m/z

40

06000 12000m/z

Sig

nal i

nten

sity

(a.

u.)50 mM ammonium citrate, pH 9.0

1000

07000 9000 120006000 8000 10000 11000

100 nM PL-10k

Laser : 120 J

Sig

nal i

nten

sity

(a.

u.)

m/z

Pullulan is a polysaccharide polymer consisting of maltotriose units.

10 M PL-6k

10 M PL-10k

Polydispersity indices Polydispersity indices (PDIs) (PDIs)

PL-6k : 1.03 (1.09 was determined from GPC)

PL-10k : 1.05 (1.10 was determined from GPC)

PDI = Mw/Mn

Mn: Number-average molecular weights

Mw: Weight-average molecular weights

Mn = (NiMi) / Ni

Mw = (NiMi2) / (NiMi)

LR R2 LODs (nM)PL-6k 0.01–5 0.9919 8.5

PL-10k (Mw 7353) 0.05–10 0.9931 28

PL-10k (Mw 9765) 0.1–10 0.9924 100

Mw 7353 764 (5.2%) 798 (7.6%)

Mw 9765 420 (10.7%) 421 (11.9%)

Mean (RSD) Mean (RSD)Intradaya Interdayb

a: n=6 ; b: n=3

Linear range & LODs Linear range & LODs

ReproducibilityReproducibility

0

2000

4000

6000

Inte

ns.

[a.

u.]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

m/z = 162Sig

nal i

nten

sity

(a.

u.)

m/z50 mM ammonium citrate, pH 9.0, 3x HgTe

Dextran (19500)

m/z ~ 12,000

Analyses of PEG–Au NPs Through HgTe NMs/SALDI-MS and Monitor Biointeractions

This method also facilitated the analyses of various functional-PEG–modified Au NPs without the need for any sample pretreatment and monitoring biointeractions .

NH2-PEG-Au NPs CM-PEG-Au NPs

Biotin-PEG-Au NPsavidin (5.0 nM)Biotin-PEG-Au NPs

Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c Through Internal

Standard Mass Spectrometry

Tryptic digest of Cyt c (500 nM) microwave irradiation for 15 s.

LOD: nM

ConclusionConclusion

HgTe are useful for small and large analytes, while AuNPs are only good for small analytes.

Internal standard MS methods allow quantitation of analytes.

MS using HgTe allows detection of proteins and their complexes with other proteins.

MS using HgTe allows determination of MW of polysaccharides.

Detect Proteins and Protein-Protein Complexes

IgG (~150 kDa, pI~5.8) &

protien G (~26 kDa, pI~4.2)

α1-antirypsin (~50 kDa, pI~5)

trypsin (~23 kDa, pI~10.5)- 2 - 4 mg/mL in blood

- 9.5~12.5 mg/ml

Brij 76 (polyoxyethylene(10) stearyl ether)

OOH

10

Anal. Chem. 2012, 84, 1924-1930.

Preparation and Characterization of Au NPs/MGs

Scheme : Microwave-accelerated enzymatic digestion of Cyt c using TR/Au NPs/MGs