Chapter 6
A Primer and Guidelines for Shotgun Proteomic Analysis
in Non-model Organisms
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
Abstract
During the last decade, we have witnessed outstanding advances in proteomics led mostly by great
technological improvements in mass spectrometry field allowing high-throughput production of highquality data used for massive protein identification and quantification. From a practical viewpoint, these
advances have been mainly exploited in research projects involving model organisms with abundant
genomic and proteomic information available in public databases. However, there is a growing number
of organisms of high interest in different disciplines, such as ecological, biotechnological, and evolutionary
research, yet poorly represented in these databases. Important advances in massive parallel sequencing
technology and easy accessibility of this technology to many research laboratories have made nowadays possible to produce customized genomic and proteomic databases of any organism. Along this line, the use of
proteogenomic approaches by combining in the same analysis the data obtained from different omic levels
has emerged as a very useful and powerful strategy to run shotgun proteomic experiments specially focused
on non-model organisms. In this chapter, we provide detailed procedures to undertake shotgun quantitative proteomic experiments following either a label-free or an isobaric labeling approach in non-model
organisms, emphasizing also a few key aspects related to experimental design and data analysis.
Key words Label-free, Isobaric labeling, Quantitative proteomics, Proteogenomics, Pooling samples,
Data normalization, Multiple testing correction methods
1 Introduction
In the last two decades, the paradigmatic scheme of molecular
biology studies, traditionally based on the analysis of a limited
number of genes, transcripts, and proteins, has evolved toward
the massive study of genes (genome), transcripts (transcriptome),
and proteins (proteome), which has expanded our possibilities to
test both new and classical hypotheses from a new perspective [1–
3]. An interesting common feature among different omic analyses is
that there is no requirement for any prior knowledge about the
biological phenomenon under study, which has significantly propelled discovery-driven research. Studies following an exploratory
research strategy can be very useful as far as new hypotheses and
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
77
A Primer and Guidelines for Shotgun Proteomic Analysis
in Non-model Organisms
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
Abstract
During the last decade, we have witnessed outstanding advances in proteomics led mostly by great
technological improvements in mass spectrometry field allowing high-throughput production of highquality data used for massive protein identification and quantification. From a practical viewpoint, these
advances have been mainly exploited in research projects involving model organisms with abundant
genomic and proteomic information available in public databases. However, there is a growing number
of organisms of high interest in different disciplines, such as ecological, biotechnological, and evolutionary
research, yet poorly represented in these databases. Important advances in massive parallel sequencing
technology and easy accessibility of this technology to many research laboratories have made nowadays possible to produce customized genomic and proteomic databases of any organism. Along this line, the use of
proteogenomic approaches by combining in the same analysis the data obtained from different omic levels
has emerged as a very useful and powerful strategy to run shotgun proteomic experiments specially focused
on non-model organisms. In this chapter, we provide detailed procedures to undertake shotgun quantitative proteomic experiments following either a label-free or an isobaric labeling approach in non-model
organisms, emphasizing also a few key aspects related to experimental design and data analysis.
Key words Label-free, Isobaric labeling, Quantitative proteomics, Proteogenomics, Pooling samples,
Data normalization, Multiple testing correction methods
1 Introduction
In the last two decades, the paradigmatic scheme of molecular
biology studies, traditionally based on the analysis of a limited
number of genes, transcripts, and proteins, has evolved toward
the massive study of genes (genome), transcripts (transcriptome),
and proteins (proteome), which has expanded our possibilities to
test both new and classical hypotheses from a new perspective [1–
3]. An interesting common feature among different omic analyses is
that there is no requirement for any prior knowledge about the
biological phenomenon under study, which has significantly propelled discovery-driven research. Studies following an exploratory
research strategy can be very useful as far as new hypotheses and
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
77