1. Analysis of Biomolecules through Nanomaterials Based Mass Spectrometry
Huan-Tsung Chang
Department of Chemistry
National Taiwan University
07, 15, 2015

2. Sensors (Abs, Fluor., and SERS)
Optical
Solar cells
Optical
Catalytic Activity
Fuel cells
Sensors (Abs, Fluor., and SERS)
Energy and Environment
Analytical Chemistry
Decontaminants
Biocompatible, Stable, and Low Cost
Cell Imaging
Antibacterial Agents
Health
Drugs

3. Large analytes peptides, proteins, ONDs, polysaccharides
Nanomaterials are used as the matrix in MS.
High absorption coefficient
Concentration effect
More than thousand (at least 10X) molecules per NPs
Easy sample preparation
Minimum problems associated with sweet spots
Small analytes
ATP, thiols,
melamine,
carbohydrates
Large analytes peptides, proteins, ONDs, polysaccharides
Cell imaging

4. Gold nanoparticles as a matrix for the quantitation
of thiol compounds
(A) Au hν
m/z
Mixtures
(B) Au hν
m/z
: GSH, Cys, and HCys;
: Arg

5. Calibration Curves Linear Range Limit of Detection [GSH + Na]+: 330.0710 20 30 40 50 60 80 70 90
100
120
140
160
R2 = 0.997
R2 = 0.996
R2 = 0.995
(A)
(B)
(C)
Calibration Curves
[GSH + Na]+:
(B) [Cys + H]+:
(C) [HCys + H]+:
Linear Range
Signal Intensity (a. u.)
GSH : 2.5 －50 mM
Hcys : 2.5 －100 mM
Cys : 5.0 －75 mM
Limit of Detection
GSH :1.0 mM
Hcys : 2.0 mM
Cys : 1.3 mM
Concentration (mM)

6. Concentration effect Signal Intensity (a. u.) LOD: nM level m/z (A)1
(A)
20 μL
1.0 mM GSH
341.26
330.07 1
(B)
0.1 mM GSH
Signal Intensity (a. u.) 1
(C)
1 mL
341.26
LOD: nM level
0.1 mM GSH
330.07
346.03
325
330
335
340
345
350
A-B: (NR)Au NPs (3) as matrices without concentration
C: 0.1 (NR)Au NPs -- concentration factor of 50
m/z

7. Detection of ATP using Apt-AuNPs
(NH4)3citrate Au
Illustration of the interactions of ATP with Apt-AuNPs and AuNPs. Au Au hν
m/z
: ATP-aptamer
: ATP

8. Determination of ATP and GSH in Red Blood Cells
1.9 (± 0.3) mM (n = 3)
Apt-AuNP + AuNP
ATP
DHB
0.94 (± 0.06) mM (n = 3)
Apt-AuNP + AuNP
GSH
DHB

9. Improved quantitation of CAP using 4-MBA as an
internal standard
A drug for treatment of hypertension
CAP:
CAP
4-Mercaptobenzoic acid
(4-MBA)
4-MBA
N-2-mercaptopropionylglycine (MPG) modified Au NPs

10. Linearity: 2.5–25 μM LOD: 1 uM 50 22 200 5.7 18 [MBA － H]－ 8.3
[CAP]
(mM)
With MBA-Au NPs (n = 3)
Without MBA-Au NPs (n = 3)
Found ± SD
Recovery
(%)
RSD
(uM) 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
Linearity:
2.5–25 μM
LOD: 1 uM
[CAP － H]－
SALDI Mass spectrum of CAP, MBA and 14-nm Au NPs (7.5 nM) prepared in 0.1 mM ammonium citrate (pH 6.00).

11. Absorbed light over UV-Vis range
Detection of Proteins through MS using HgTe
UV-Vis
Energy-dispersive X-ray (EDX)
Existence of
HgTe
Absorbed light over UV-Vis range
5 mM BSA
SA as matrix
Dominated peak:
[M + H]+
EDX:當原子的內層電子受到外來能量源 (如：電子束、離子束或者光源等) 的激發而脫離原子時，原子的外層電子將很快的遷降至內層電子的空穴並釋放出兩能階差能量。被釋出的能量可能以 X光的形式釋出，或者此釋出的能量將轉而激發另一外層電子使其脫離原子。
Laser shots : 300; laser pulse energy :62.5 μJ (power density :2 × 109 W/cm2).
LOD: 14 nM

12. Mass limit : IgG (m/z ~ 150 kDa)
Quasimolecular ions of two identical
light chains of IgG
HgTe has a low melting temperature and low thermal conductivity.
Generation of higher temperature can be achieved for efficient desorption/ionization of the analytes.
150 kDa ions detected 12
IgG (5 mM)

13. Detection of protein-protein complexes
Enhance 6-fold
w/o Brij 76
w/ 1% Brij 76
5 uM a1-antitrypsin
IgG
w/o Brij 76
1.7 uM trypsin
protein G
w/ 0.1% Brij 76
IgG + protein G
5 uM a1-antitrypsin uM trypsin
w/ 0.1% Brij 76
a1-antitrypsin: 3 uM (1.5 pmol)
Trypsin: 0.5 uM (0.25 pmol)
IgG: 5 uM (2.5 pmol)
protein G: 2 uM (1 pmol)
LOD:

14. Detection of carbohydrates in vrious honey samples
Longan honey
Maltose
Fructose
LODs (from standard solutions)
Fructose: 15 μM
Maltose: 10 μM

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

16. pomelo honey
m/z 650–2700 Da
Oligosaccharides?

17. SALDI mass spectra of sucrose, -CD, -CD, and -CD
[Sucrose + Na]+
365.3
400
3000
340
8000
995.8
[-CD + Na]+
Signal intensity (a. u.)
970
1030
4000
1320.3
996.1
[-CD + Na]+
1158.0
[-CD + Na]+
[-CD + Na]+
900
1400
m/z 17
0.5 mM ammonium citrate, pH mM NaCl, 1X HgTe

18. Pullulan is a polysaccharide polymer consisting of maltotriose units.
5893.9
5406.6
6377.4
4919.7
4432.3
6861.1
m/z
100
4000
8000
Signal intensity (a. u.)
5000
6000
7000
0.5 mM ammonium citrate, pH 7.0
7347.5
1500
100 nM PL-6k
Laser : 75 J
Matrix : 1x HgTe
7838
8321
8803
9285
9767
7356
6871
10248
10727
6387
11211 40
12000
50 mM ammonium citrate, pH 9.0
1000
9000
10000
11000
100 nM PL-10k
Laser : 120 J
10 M
PL-6k
10 M
PL-10k

19. Polydispersity indices (PDIs)
PDI = Mw/Mn
Mn: Number-average molecular weights
Mn = (NiMi) / Ni
Mw: Weight-average molecular weights
Mw = (NiMi2) / (NiMi)
PL-6k : 1.03 (1.09 was determined from GPC)
PL-10k : 1.05 (1.10 was determined from GPC)

20. Linear range & LODs Reproducibility LR R2 LODs (nM)
PL-6k –
PL-10k (Mw 7353) –
PL-10k (Mw 9765) –
Reproducibility
Mw (5.2%) (7.6%)
Mw (10.7%) (11.9%)
Mean (RSD) Mean (RSD)
Intradaya Interdayb
a: n=6 ; b: n=3

21. Dextran (19500) Signal intensity (a. u.) m/z m/z = 162 m/z ~ 12,000
50 mM ammonium citrate, pH 9.0, 3x HgTe

22. Analyses of PEG–Au NPs Through HgTe NMs/SALDI-MS and Monitor Biointeractions
 CM-PEG-Au NPs
NH2-PEG-Au NPs
Biotin-PEG-Au NPs
Biotin-PEG-Au NPs
avidin (5.0 nM)
that monitoring biointeractions on nanomaterials is relatively straightforward when using our developed SALDI-MS approach to detect the ligand’s signal.
This method also facilitated the analyses of various functional-PEG–modified Au NPs without the need for any sample pretreatment and monitoring biointeractions .

23. Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c Through Internal Standard Mass Spectrometry

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

25. LOD: nM

26. Conclusion
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.

27. Thank you for your attention

28. Detect Proteins and Protein-Protein Complexes
IgG (~150 kDa, pI~5.8) &
protien G (~26 kDa, pI~4.2)
9.5~12.5 mg/ml
α1-antirypsin (~50 kDa, pI~5)
trypsin (~23 kDa, pI~10.5)
 mg/mL in blood
Brij 76 (polyoxyethylene(10) stearyl ether)
Anal. Chem. 2012, 84,

29. Scheme : Microwave-accelerated enzymatic digestion of Cyt c using TR/Au NPs/MGs
Preparation and Characterization of Au NPs/MGs