Chapter 16
Shotgun Proteomics and Mass Spectrometry as a Tool
for Protein Identification and Profiling of Bio-Carrier-Based
Therapeutics on Human Cancer Cells
Syafiq Asnawi Zainal Abidin, Iekhsan Othman, and Rakesh Naidu
Abstract
Shotgun proteomics has been widely applied to study proteins in complex biological samples. Combination
of high-performance liquid chromatography with mass spectrometry has allowed for comprehensive
protein analysis with high resolution, sensitivity, and mass accuracy. Prior to mass spectrometry analysis,
proteins are extracted from biological samples and subjected to in-solution trypsin digestion. The digested
proteins are subjected for clean-up and injected into the liquid chromatography-mass spectrometry system
for peptide mass identification. Protein identification is performed by analyzing the mass spectrometry data
on a protein search engine software such as PEAKS studio loaded with protein database for the species of
interest. Results such as protein score, protein coverage, number of peptides, and unique peptides identified
will be obtained and can be used to determine proteins identified with high confidence. This method can be
applied to understand the proteomic changes or profile brought by bio-carrier-based therapeutics in vitro.
In this chapter, we describe methods in which proteins can be extracted for proteomic analysis using a
shotgun approach. The chapter outlines important in vitro techniques and data analysis that can be applied
to investigate the proteome dynamics.
Key words Shotgun proteomics, Protein identification, Protein profiling, Bio-carrier vectors
1 Introduction
1.1 Proteomics
and Mass
Spectrometry
Proteomics is defined as a large-scale analysis of proteome; a set of
proteins produced in an organism, a system, or any biological
context. The study of proteomics aimed to identify and quantify
proteins in a proteome which encompasses the expression, localization, interactions, and post-translational modifications [1]. Proteomics has been described as an important approach to obtain
biological information since most biological activities are attributed
by proteins, thus enhancing our conception of a biological system
[2]. Advancement of proteomics study has been driven by the
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
233
Shotgun Proteomics and Mass Spectrometry as a Tool
for Protein Identification and Profiling of Bio-Carrier-Based
Therapeutics on Human Cancer Cells
Syafiq Asnawi Zainal Abidin, Iekhsan Othman, and Rakesh Naidu
Abstract
Shotgun proteomics has been widely applied to study proteins in complex biological samples. Combination
of high-performance liquid chromatography with mass spectrometry has allowed for comprehensive
protein analysis with high resolution, sensitivity, and mass accuracy. Prior to mass spectrometry analysis,
proteins are extracted from biological samples and subjected to in-solution trypsin digestion. The digested
proteins are subjected for clean-up and injected into the liquid chromatography-mass spectrometry system
for peptide mass identification. Protein identification is performed by analyzing the mass spectrometry data
on a protein search engine software such as PEAKS studio loaded with protein database for the species of
interest. Results such as protein score, protein coverage, number of peptides, and unique peptides identified
will be obtained and can be used to determine proteins identified with high confidence. This method can be
applied to understand the proteomic changes or profile brought by bio-carrier-based therapeutics in vitro.
In this chapter, we describe methods in which proteins can be extracted for proteomic analysis using a
shotgun approach. The chapter outlines important in vitro techniques and data analysis that can be applied
to investigate the proteome dynamics.
Key words Shotgun proteomics, Protein identification, Protein profiling, Bio-carrier vectors
1 Introduction
1.1 Proteomics
and Mass
Spectrometry
Proteomics is defined as a large-scale analysis of proteome; a set of
proteins produced in an organism, a system, or any biological
context. The study of proteomics aimed to identify and quantify
proteins in a proteome which encompasses the expression, localization, interactions, and post-translational modifications [1]. Proteomics has been described as an important approach to obtain
biological information since most biological activities are attributed
by proteins, thus enhancing our conception of a biological system
[2]. Advancement of proteomics study has been driven by the
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
233