Mass spectrometry (MS) proves to be one of the more sensitive techniques in proteomics which, while making it an incredibly useful technology, leaves it susceptible to strict sample preparation requirements. The conditions of any particular MS experiment will ultimately dictate the degree and types of preparations necessary but some concepts can be applied to any MS experiment, such as enrichment and fractionation. Furthermore, with the current emphasis being put on high-throughput techniques involving MS there is an increasing demand for automated and streamlined sample preparation tools for protein and peptide extraction upstream of MS.
The first step to any proteomics experiment, particularly MS, is often to reduce the complexity of the sample; or fractionate the sample. The goal of fractionation is usually to remove the “highly abundant components of the proteome”[1] followed by “subsequent fractionation of the moderate to low abundance proteins”[1] in order to produce a concentrated sample of proteins with the potential to be clinically relevant. There are a number of fractionation protocols and tools available and it is not uncommon to utilize more than one to adequately reduce the convolution of a sample. A protein sample can be fractionated on the basis of size, charge, hydrophobicity, and/or binding affinity. These qualities are often the basis of the many available kits capable of enriching a sample by partitioning out highly abundant proteins from a sample such as serum. Examples of such kits include Beckman Coulter’s ProteomeLab IgY-12 spin and LC column kits which “selectively partition 12 highly abundant proteins found in serum or plasma”[1] or Bio-Rad’s ReadyPrep protein extraction kits which “fractionate protein mixtures based on cellular location (the cytoplasmic/nuclear fraction, membrane proteins, and membrane proteins specifically involved in intracellular signaling).”[1]
The clean up of a protein sample is more crucial prior to introduction to the mass spectrometer than in most other proteomics experiments primarily because of the sensitivity of the technology, but also because ‘dirty samples’ can be quite detrimental to the machine.
In terms of MS sensitivity, it is “necessary to remove the detergents, [ion suppressing] salts, and chaotropes commonly used in proteomic sample preparation to increase solubility”[1] as they can compromise analysis. In cleaning up samples for MS, one can utilize any number of concepts described in the Contaminant Elimination section of this chapter. Finally, it should be noted that it is important to maintain an awareness of protein yield while attempting to maximize the quality of data.[1]
2. Optimization of Proteomic Sample Preparation Procedures for Comprehensive Protein Characterization of Pathogenic Heather M. Mottaz-Brewer, Angela D. Norbeck, Joshua N. Adkins, Nathan P. Manes, Charles Ansong, Liang Shi, Yasuko Rikihisa, Takane Kikuchi, Scott W. Wong, Ryan D. Estep, Fred Heffron, Ljiljana Pasa-Tolic, and Richard D. Smith=Systems(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2628077&tool=pmcentrez)
Heather M. Mottaz-Brewer, Angela D. Norbeck, Joshua N. Adkins, Nathan P. Manes, Charles Ansong, Liang Shi, Yasuko Rikihisa, Takane Kikuchi, Scott W. Wong, Ryan D. Estep, Fred Heffron, Ljiljana Pasa-Tolic, and Richard D. Smith
J Biomol Tech. 2008 December; 19(5): 285–295.
PMCID: PMC2628077
Main Focus
The results that can be derived from an experiment depends on the way the sample used was prepared. Optimizing the steps in sample preparation to get better results in mass spectrometry can help in the better understanding of host pathogen interactions.
Summary
Solid Phase Extraction Manifold
Mass Spectrometry can be used in proteomics to find out about the interactions pathogens have with the host. However in any analysis the results are largely dependent on the characteristics of the sample used. Changing the way the sample was prepared in the experiment can lead to better results. There are a lot of different steps in sample handling that can be optimized to give better results. However Influence diagrams need to be made to determine the changes made to the sample preparation procedures. For example an increase in selectivity leads to a decrease in sensitivity and vice versa. Biomass losses have to be considered as well. The different steps which can be optimized are:
Proteins of interest – The protein of interest should be identified and its possible location found out. The experiment should be designed keeping this in mind.
Growth conditions – The growth conditions that are most suitable for the particular host pathogen can be designed
Sample Preparation – Sample size used and the way it is prepared can be changed to optimized for each experiment
Sample Analysis Platforms – The platform used to analyze the samples should be considered
Data Analysis – It is important how the data found out can be analyzed. Since not every bacterium study has been sequenced optimal data analysis plays an important role.
Tweaking the different steps above can lead to better results in finding out the interactions between pathogens and hosts leading to drug discovery.
New Terms
HeLa cell
Immortal cell line used in research, which are derived from cervical cancer cells of Henrietta Lacks( http://en.wikipedia.org/wiki/HeLa )
BCA protein assay
Bicinchoninic acid assay is an assay used to determine the total level of protein or the protein concentration in a solution. The extent of color change from green to purple indicates how much protein is present.
It is a compact graphical and mathematical representation of a decision situation. It is a generalization of a Bayesian networking which not only probabilistic inference problems but also decision making problems (following maximum expected utility criterion) can be modeled and solved.(http://en.wikipedia.org/wiki/Influence_diagram)
Lipidomics
Study of pathways and networks of cellular lipids.
Course Relevance
This paper discusses the importance sample preparation and sample analysis has in the analysis of experiments. It tells us the different optimizations we can perform in various steps to get a better result.
The different sample preparation kits and reagents that are available and can be used to optimize mass spectrometry results
Summary
The website talks about the different reagents and kits that can be used to optimize sample preparation. The different kits available are:
SILAC Kits and Reagents - Kit used for stable isotope labeling using amino acids in cell culture. It can quantify and identify relative differential changes in protein sample
Tandem Mass Tag Technology - Isobaric chemical tags that enable concurrent identification and quantification of proteins in different samples.
Use of deuterated crosslinkers
2D sample preparation kits and albumin removal kits form optimizing sample preparation
SilverSNAP stain for silver staining of 2D gels to increase sensitivity
Strong Ion Exchange spin columns for easy and better purification
Trifluoroacetic acid for reverse phase peptide separations
Formic Acid ampules as a contamination free alternative for preparing elution solvents for HPLC separation of proteins and peptides
PepClean C18 Spin columns for purifying or concentrating multiple peptide samples in less than 30 minutes
Protein Precipitation plates for target molecule isolation
Phosphopeptide isolation kit for isolating phosphorylated peptides from complex protein digests
Matrices for MALDI Analysis
New Terms
SILAC (Stable isotope labelling with amino acids in cell culture)
A mass spectrometry-based technique developed to detect differences in protein abundance between two (or more) samples( http://en.wikipedia.org/wiki/SILAC )
Deuterated crosslinkers
Chemical reagents which when used in mass spec studies produces a Dalton shift compared to proteins cross linked with the non deuterated analog. The two analogs can be used for isotopically labeling proteins and peptides.
Elution
The emergence of a chemical from a chromatographic column.
MALDI
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods(http://en.wikipedia.org/wiki/MALDI-TOF)
Course Relevance
The website details the different tools and reagent kits that one can use to optimize the sample preparation in the proteomics laboratory
18O Stable Isotope Labeling in MS-based Proteomics
Xiaoying Ye, Brian Luke, Torkell Andresson and Josip Blonder. Briefings in Functional Genomics and Proteomics. Vol 8. NO 2. 136-144.
http://www.ncbi.nlm.nih.gov/pubmed/19151093
This article is a review of a mass spectrometry(MS) labeling technique using 18O isotopes for proteomics research. The article gives a robust background of the history and principles of the technique and then goes on to explain different specific technique applications. The article then concludes with an evaluation of the circumstances when it is best practice. Areas the versatile technique is applied to include: global proteomic investigations, targeted proteomic investigations, and post-translational modifications.
18O stable isotope labeling in the context of mass spectrometry is a technique that is used in many assay protocols. In simplest terms, the stable oxygen isotope 18O is attached to the target protein in an attempt to identify and quantify unknown proteins. Details vary from there depending on the goal of the experiment. The article identifies details of common methods and the categories of the results produced. One group of methods uses the metabolic function of the cell or organism to insert the isotope. Other methods label using chemical or enzymatic incorporation to accomplish the binding of the isotope.
While not the most common isotope labeling technique 18O labeling has certain advantages. Results are generated very quickly, the materials needed to run the assay are relatively cheap, and the amount of sample needed is small. This third point is important as it allows using amount-limited samples, such as those obtained through clinical methods, to be used effetely.
Global proteomic investigations: using 18O labeling samples are profiled quantitatively on a proteome wide basis. Problems can occur through variable cleavage causing error in results. However an unbiased coverage of the proteome is a major advantage over 2D-PAGE-based methods.
Targeted proteomic investigations: techniques using 18O labeling to identify proteins in low abundance. Useful in early diction of biomarkers for disease or identifying proteins used in sorting and trafficking in cell organs.
Post-translational modifications: using 18O labeling to identify proteomes used in process regulation. Quantifying the abundance of the proteome before, after and after translation.
Compared to other technique: 18O labeling has certain advantages making it very useful under the right conditions. Reagents are relatively cheaper than other assay techniques. Assay turn around times is short. This is useful for generating quick results needed in clinical settings. Assays can be conducted with small sample volumes. Using less sample is very important when dealing with limited supply samples.
Conclusion, the choice of technique depends on experimental design. While not always the best choice, 18O labeling is very efficient at generating certain types of information quickly and inexpensively.
This article was an overview of the diverse application of the 18O labeling technique. It sets forth a very thorough background of the technique and principle. Sections are then presented on major areas where 18O labeling has and continues to be applied. Breaking down the history of the technique, improvements and breakthroughs in major research areas.
