1.2 Bio-Carriers
and Proteomics
The advancements of bio-carrier technology have been proven to
be crucial in developing effective therapeutic agents against various
diseases. Nanotechnology plays a key role in bio-carrier development by efficiently delivers drug to the target site with minimum
side effects and improved drug reaction [8]. To date, various types
of nano-sized bio-carrier (nanoparticles) and their therapeutic
applications have been extensively reviewed [8–10]. In this chapter,
the methodology to understand the proteomic changes in cell
and/or tissue cultures upon interaction with bio-carrier-based
drug is discussed.
2 Materials
2.1 Protein Samples,
Extraction,
and Quantification
1. Lyophilized protein samples (see Note 1).
2. Tissue homogenizer.
3. RIPA Lysis and Extraction Buffer (ThermoFisher, USA) with
1Â Protease and Phosphatase inhibitor.
4. Pierce BCA Protein Assay Kit (ThermoFisher, USA).
2.2 Trypsin Digestion
(In-Solution Protein
Digestion)
1. Trypsin stock (20 μg of lyophilized trypsin, MS grade): Add
20 μL of 50 mM acetic acid to 20 μg of lyophilized trypsin.
2. 1.5 mL Protein Lo-bind centrifuge tubes (Eppendorf).
3. Trifluoroethanol (TFE).
4. Digestion buffer: 100 mM ammonium bicarbonate (ABC).
5. Reducing buffer: 200 mM dithiothreitol (DTT).
6. Alkylation buffer: 200 mM iodoacetamide (IAM).
7. Formic acid, MS grade.
8. Vacuum concentrator.
2.3 Peptide
Concentration
and Desalting
1. Pierce C18 Spin Column (ThermoFisher, USA).
2. 1.5 mL Protein Lo-bind centrifuge tubes.
3. Solutions (see Note 2).
(a) Activation solution: 50% acetonitrile (ACN).
(b) Equilibration solution: 0.5% formic acid (FA) in 5% ACN.
(c) Wash solution: 0.5% FA in 5% ACN.
(d) Sample buffer: 2% FA in 20% ACN (3 parts sample to
1-part sample buffer).
(e) Elution buffer: 70% ACN.
2.4 Liquid
ChromatographyMass Spectrometry
(LC-MS)
1. Agilent 1200 Series Nano and Capillary System LC with Agilent 6550 iFunnel Quadrupole-Time-of-Flight (Q-TOF)
LC-MS, coupled with ChipCube nano-electrospray ionization
(nano-ESI) source (see Note 3).
Proteomics and Mass Spectrometry
235
and Proteomics
The advancements of bio-carrier technology have been proven to
be crucial in developing effective therapeutic agents against various
diseases. Nanotechnology plays a key role in bio-carrier development by efficiently delivers drug to the target site with minimum
side effects and improved drug reaction [8]. To date, various types
of nano-sized bio-carrier (nanoparticles) and their therapeutic
applications have been extensively reviewed [8–10]. In this chapter,
the methodology to understand the proteomic changes in cell
and/or tissue cultures upon interaction with bio-carrier-based
drug is discussed.
2 Materials
2.1 Protein Samples,
Extraction,
and Quantification
1. Lyophilized protein samples (see Note 1).
2. Tissue homogenizer.
3. RIPA Lysis and Extraction Buffer (ThermoFisher, USA) with
1Â Protease and Phosphatase inhibitor.
4. Pierce BCA Protein Assay Kit (ThermoFisher, USA).
2.2 Trypsin Digestion
(In-Solution Protein
Digestion)
1. Trypsin stock (20 μg of lyophilized trypsin, MS grade): Add
20 μL of 50 mM acetic acid to 20 μg of lyophilized trypsin.
2. 1.5 mL Protein Lo-bind centrifuge tubes (Eppendorf).
3. Trifluoroethanol (TFE).
4. Digestion buffer: 100 mM ammonium bicarbonate (ABC).
5. Reducing buffer: 200 mM dithiothreitol (DTT).
6. Alkylation buffer: 200 mM iodoacetamide (IAM).
7. Formic acid, MS grade.
8. Vacuum concentrator.
2.3 Peptide
Concentration
and Desalting
1. Pierce C18 Spin Column (ThermoFisher, USA).
2. 1.5 mL Protein Lo-bind centrifuge tubes.
3. Solutions (see Note 2).
(a) Activation solution: 50% acetonitrile (ACN).
(b) Equilibration solution: 0.5% formic acid (FA) in 5% ACN.
(c) Wash solution: 0.5% FA in 5% ACN.
(d) Sample buffer: 2% FA in 20% ACN (3 parts sample to
1-part sample buffer).
(e) Elution buffer: 70% ACN.
2.4 Liquid
ChromatographyMass Spectrometry
(LC-MS)
1. Agilent 1200 Series Nano and Capillary System LC with Agilent 6550 iFunnel Quadrupole-Time-of-Flight (Q-TOF)
LC-MS, coupled with ChipCube nano-electrospray ionization
(nano-ESI) source (see Note 3).
Proteomics and Mass Spectrometry
235