Proteomics investigates on a large-scale the proteins expressed in a given organism or biological system.
A top-down proteomic approach aims to identify characteristics of whole proteins, while bottom-up proteomics—also known as shotgun proteomics—uses a high throughput workflow to analyze complex mixtures containing hundreds to thousands of unique proteins.
Large amounts of proteomic data is increasingly being produced, often as a complement to genomic results.
As proteins are the functional macromolecules directly driving cellular activity and physiology, proteomics offers significant biological insights not readily addressed by genomics and transcriptomics.
These include studies in post-translational protein modifications, spatial and temporal protein localization, the biochemistry of metabolic pathways, protein interactions, and more.
Over the past few years, proteomic technologies have centered around analyses by mass spectrometry or two-dimensional gels.
However, as proteins are inherently complex in terms of stability, localization, and variation in expression, nuances in various steps of the proteomic preparation workflow warrant consideration.
Cell Lysis and Extraction
Cell lysis that precedes protein extraction can occur mechanically, through
homogenizers or sonicators
, or chemical solubilization via detergents and lysis buffers.
While mechanical disruption offers an advantage of maintaining a relatively native chemical environment, reagents-based lysis via
detergents
or
lysis buffers
are generally more accommodating to smaller sample volumes and higher throughput processing.
Ready-to-use
protein extraction kits
can offer a rapid, straightforward solution for lysing cells and subsequent protein collection.
Cell Fractionation
Depending on the aim of the study, complex samples, such as whole tissue and cell extracts, may require additional
fractionation
.
This step can ensure enrichment of proteins enriched in subcellular locations, such as
membrane proteins
or
nuclear proteins
.
Highly abundant proteins can also limit peptide identification during LC-MS analysis.
Further fractionation, through the use of centrifugation,
chromatography
or
protein concentration kits
, can help to account for the variations in protein amounts and to effectively capture lower abundance proteins.
Protein Concentration and Quantification
Protocols leading up to protein digestion often specify a certain amount of necessary sample protein.
Protein concentration kits
are available when working with dilute or low-abundance protein samples.
Such kits may serve to further cleanup the sample by desalting or buffer exchange.
For protein quantification, a variety of
protein assay kits
may be used, but be careful to consider any detergent or buffer compatibilities.
Protein Digestion
To generate peptides for mass spectrometric analysis, in-solution or in-gel
digestion
are the two common strategies.
With in-solution digestion, proteins can be denatured, digested, alkylated and reduced in a single tube, making this option ideal for less complex, detergent-sensitive, or low-volume samples.
With in-gel digestion,
protein electrophoresis
(typically SDS-PAGE) is used to first separate and denature proteins, which are then digested while embedded in a gel slice.
Gel-based digestion is widely used in LC-MS/MS analyses of complex samples.
The choice of
protease
may vary depending on the nature of sample proteins, trypsin being the most common.
Peptide Cleanup and Mass Spectrometry
As detergents and other contaminants obscure mass spectrometry readings, peptides must be cleaned prior to analysis.
The C18
reverse phase
is the most commonly used liquid chromatography stationary phase resin for cleanup and can come in column, cartridge, or pipette tip formats.
MALDI and ESI are the two choice ionization methods leading up to MS and MS/MS analysis, with LC-ESI-MS/MS systems being favored for complex samples.
Mass spectrometry
services
that specialize in proteomic analyses are also available.
Relevant Literature:
- Feist P, Hummon AB. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int J Mol Sci. 2015 Feb 5;16(2):3537-63. doi: 10.3390/ijms16023537. Review. PubMed PMID: 25664860
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- Barnouin K. Guidelines for experimental design and data analysis of proteomic mass spectrometry-based experiments. Amino Acids. 2011 Feb;40(2):259-60. doi: 10.1007/s00726-010-0750-9. Epub 2010 Oct 1. PubMed PMID: 20886359.
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