through different steps of liquid chromatography (LC) (for example, using different types of chromatographic columns sequentially;
see MudPIT system [10]) before facing massive MS analysis, an
approach known as shotgun proteomics. Sample fractionation
before MS analysis is a step needed to reduce sample complexity,
which improves resolution for protein identification and quantification. Other successful shotgun proteomic strategies have made
use of an initial separation and fractionation of proteins according
to their molecular weight (1-D SDS-PAGE), or of peptides according to their isoelectric point (isoelectrofocusing, IEF), followed by
massive LC–MS analysis of peptides resulting from in-gel protein
digestion (in the case of 1-D SDS-PAGE) [11]. Weakness and
strengths of each method have been thoughtfully discussed elsewhere [12, 13] though shotgun proteomics can be nowadays considered the workhorse for most proteomic projects. Improvements
in the quantitative shotgun proteomic analysis have been achieved
thanks to important advances in protein sample labeling and multiplexing capacity, making labeling strategy a more attractive and
reliable option for quantitative analysis than label-free approaches.
There are different protein sample labeling and multiplexing strategies. Fundamental aspects and strengths of each strategy have
been thoughtfully described and discussed elsewhere [13–
16]. One of the most used protein labeling methods is based on
the use of isobaric labels which allow the identification and relative
quantification of proteins from different samples in the same MS
run. The best known (commercial) are the isobaric tags for relative
and absolute quantification-iTRAQ (AB Sciex) and tandem mass
tags-TMT (Thermo Fisher Scientific), with different sample multiplexing capacity, from 4 to 16 multiplexed samples in each experiment/MS run. In this chapter, we will provide detailed procedures
to undertake quantitative shotgun proteomic experiments following either a label-free approach preceded by 1-D SDS-PAGE-based
fractionation or an isobaric labeling TMT-multiplex™ approach
followed by peptide fractionation in a high pH reversed-phase
spin column, in non-model organisms, highlighting a few key
aspects to be considered in the experimental design and data
analysis.
2 Materials
2.1 Total Protein
Extraction
and Quantification
1. Lysis buffer: 7 M urea, 2 M thiourea, 4% CHAPS. Weigh
10.5 g urea, 3.8 g thiourea, and 1 g CHAPS, and dissolve in
25 mL ultrapure water. Keep frozen (À80
C) in 1 mL aliquots.
2. Ice.
3. Sonicator, including an ultrasonic microtip-probe to process
small volumes (0.2–5 mL).
80
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
see MudPIT system [10]) before facing massive MS analysis, an
approach known as shotgun proteomics. Sample fractionation
before MS analysis is a step needed to reduce sample complexity,
which improves resolution for protein identification and quantification. Other successful shotgun proteomic strategies have made
use of an initial separation and fractionation of proteins according
to their molecular weight (1-D SDS-PAGE), or of peptides according to their isoelectric point (isoelectrofocusing, IEF), followed by
massive LC–MS analysis of peptides resulting from in-gel protein
digestion (in the case of 1-D SDS-PAGE) [11]. Weakness and
strengths of each method have been thoughtfully discussed elsewhere [12, 13] though shotgun proteomics can be nowadays considered the workhorse for most proteomic projects. Improvements
in the quantitative shotgun proteomic analysis have been achieved
thanks to important advances in protein sample labeling and multiplexing capacity, making labeling strategy a more attractive and
reliable option for quantitative analysis than label-free approaches.
There are different protein sample labeling and multiplexing strategies. Fundamental aspects and strengths of each strategy have
been thoughtfully described and discussed elsewhere [13–
16]. One of the most used protein labeling methods is based on
the use of isobaric labels which allow the identification and relative
quantification of proteins from different samples in the same MS
run. The best known (commercial) are the isobaric tags for relative
and absolute quantification-iTRAQ (AB Sciex) and tandem mass
tags-TMT (Thermo Fisher Scientific), with different sample multiplexing capacity, from 4 to 16 multiplexed samples in each experiment/MS run. In this chapter, we will provide detailed procedures
to undertake quantitative shotgun proteomic experiments following either a label-free approach preceded by 1-D SDS-PAGE-based
fractionation or an isobaric labeling TMT-multiplex™ approach
followed by peptide fractionation in a high pH reversed-phase
spin column, in non-model organisms, highlighting a few key
aspects to be considered in the experimental design and data
analysis.
2 Materials
2.1 Total Protein
Extraction
and Quantification
1. Lysis buffer: 7 M urea, 2 M thiourea, 4% CHAPS. Weigh
10.5 g urea, 3.8 g thiourea, and 1 g CHAPS, and dissolve in
25 mL ultrapure water. Keep frozen (À80
C) in 1 mL aliquots.
2. Ice.
3. Sonicator, including an ultrasonic microtip-probe to process
small volumes (0.2–5 mL).
80
Angel P. Diz and Paula Sa ´ nchez-Marı ´n