Immunoglobulin G (IgG) – the second most abundant protein in human blood plasma and extra cellular fluid – plays a vital role in the immune system. Moreover, this protein has been in demand for many medical applications. For this reason, development of analytical methods using modern instruments for better understanding of IgG is becoming one of the greatest interests of researchers. Herein, a quantitative mass analysis technique using nanodiamond (ND)-assisted sample preparation for matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) is proposed. The method allows the direct detection of IgG with concentration as low as ~2 nM. It demonstrated a high reproducibility of different matrix-analyte crystal thus, verifying its applicability for quantitation. The study further aims to use the quantitation technique to compare the maximum loading capacities of untreated and differently treated carboxylated/oxidized NDs. It was found out that despite having a lot of similar properties air-oxidized ND (aoND) and commercially obtained carboxylated ND (cND) have different maximum loading capacity: ~50 nM and ~ 27 nM respectively.

Acknowledgements ………………………………………………………………………….. i
Abstract ……………………………………………………………………………………… ii
Table of Contents ……………………………………………………………………………. iii
Index of Figures ……………………………………………………………………………... iv
Tables ………………………………………………………………………………………... v
Chapter 1: Introduction ………………………………………………………………………. 1
1.1. Quantitation Methods for Protein (Immunoglobulin G) ………………………………. 1
1.2. MALDI-TOF MS application in protein analysis ……………………………………... 3
1.2.1. MALDI-TOF MS with nanodiamond (ND) …………………………………….. 4
1.3. Research Motivation …………………………………………………………………... 6
Chapter 2: Experimental Section …………………………………………………………….. 8
2.1. Preparation of ND samples for surface characterization ……………………...……….. 8
2.1.1. FTIR measurement ……………………………………………………………… 8
2.1.2. Raman Spectroscopy ……………………………………………………………. 9
2.1.3. Particle size and zeta potential measurement …………………………………… 9
2.2. IgG sample preparation ………………………………………………………………... 9
2.3. MALDI sample preparations …………………………………………………………... 9
2.3.1. Matrix …………………………………………………………………………… 9
2.3.2. IgG-ND complex ……………………………………………………...……….. 10
2.3.3. MALDI-TOF mass analysis …………………………………………………… 10
2.4. Microscope Imaging …………………………………………………………………… 10
Chapter 3: Results and Discussion …………………………………………………………... 11
3.1. Characterization of ND surfaces ……………………………………………………… 11
3.1.1. FTIR spectroscopy …………………………………………………………….. 11
3.1.2. Raman spectroscopy and
size distribution measurement …………………………………………………. 12
3.1.3. Physical adsorption of IgG on ND surfaces ……………………………………. 15
3.2. Mass spectra with/without diamond particles ………………………………………... 16
3.3. Shot to shot (within the spot) signal intensity variation of different
matrix crystals formed with ND …………………………………………………...…. 17
3.4. Quantitation analysis with MALDI-TOF MS ………………………………………... 18
Chapter 4: Conclusion ………………………………………………………………………. 28
References …………………………………………………………………………………... 29
Appendix A: Protein to ND ratio ……………………………………………………………. 32

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