Limits...
Proteomic solutions for analytical challenges associated with alcohol research.

MacCoss MJ, Wu CC - Alcohol Res Health (2008)

Bottom Line: Proteins do not conform to any one uniform sample preparation method and/or biochemical analysis.Furthermore, because the number of biological replicates involved in behavioral analyses typically is high, robust high-throughput proteomic platforms will be required to handle the multitude of protein samples that can potentially result from the various brain regions for the numerous animal models and paradigms.Finally, these effects often are monitored over time courses, again inflating the total number of samples that need to be analyzed and compared.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences at the University of Washington, Seattle, Washington.

ABSTRACT
Alcohol addiction is a complex disease with both hereditary and environmental influences. Because molecular determinants contributing to this phenotype are difficult to study in humans, numerous rodent models and conditioning paradigms have provided powerful tools to study the molecular complexities underlying these behavioral phenotypes. In particular, specifically bred rodents (i.e., selected lines and inbred strains) that differ in voluntary alcohol drinking represent valuable tools to dissect the genetic components of alcoholism. However, because each model has distinct advantages, a combined comparison across datasets of different models for common changes in gene expression would provide more statistical power to detect reliable changes as opposed to the analysis of any one model. Indeed, meta-analyses of diverse gene expression datasets were recently performed to uncover genes related to the predisposition for a high alcohol intake. This large endeavor resulted in the identification of 3,800 unique genes that significantly and consistently changed between all included mouse lines and strains . Similar experiments also are crucial at the protein level. However, these analyses are not yet possible. Proteins do not conform to any one uniform sample preparation method and/or biochemical analysis. They display a broad range of physical and chemical properties (e.g., molecular weight or hydrophobicity) and are expressed over a very large dynamic range (up to 8 orders of magnitude). Further complicating global proteomic comparisons are the added considerations that proteins often undergo extensive covalent modifications and that protein functions often are regulated by complex protein-protein interactions and the specific location of the proteins in the cell (i.e., their subcellular localization)). Furthermore, because the number of biological replicates involved in behavioral analyses typically is high, robust high-throughput proteomic platforms will be required to handle the multitude of protein samples that can potentially result from the various brain regions for the numerous animal models and paradigms. Finally, these effects often are monitored over time courses, again inflating the total number of samples that need to be analyzed and compared. This article summarizes some general strategies for large-scale, high-throughput protein analyses and describes two new proteomic strategies that appear promising for future studies in this field.

Show MeSH

Related in: MedlinePlus

Illustration of selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. A) A predetermined precursor ion (mass-to-charge ratio, m/z 521.7) is selected in the first mass analyzer (Q1), fragmented by collision-induced dissociation (CID), and one of the resultant product ions (m/z 757.6) is selectively monitored in the second mass analyzer (Q3). B) Example of four different peptides generated by protein digestion with the enzyme trypsin (i.e., tryptic peptides) measured by SRM in human plasma.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3860482&req=5

f10-arh-31-3-251: Illustration of selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. A) A predetermined precursor ion (mass-to-charge ratio, m/z 521.7) is selected in the first mass analyzer (Q1), fragmented by collision-induced dissociation (CID), and one of the resultant product ions (m/z 757.6) is selectively monitored in the second mass analyzer (Q3). B) Example of four different peptides generated by protein digestion with the enzyme trypsin (i.e., tryptic peptides) measured by SRM in human plasma.

Mentions: Using the selectivity of multiple stages of mass selection of a tandem mass spectrometer (see figure 10), these targeted SRM assays are the mass spectrometry equivalent of a Western blot.5 In fact, Arnott and colleagues (2002) originally coined the term “MS Westerns” for the use of tandem mass spectrometry to target individual hypothesized peptides. Just as a Western blot only produces a signal for proteins within a complex mixture that are recognized by an antibody, a targeted mass spectrometry assay will only produce a signal for peptides that have a specific combination of precursor and product ion m/z. This combination of precursor and product ion m/z is extremely selective and referred to as a SRM transition. The advantage of using a targeted mass spectrometry–based assay is that it does not require creating any immunoaffinity reagents.


Proteomic solutions for analytical challenges associated with alcohol research.

MacCoss MJ, Wu CC - Alcohol Res Health (2008)

Illustration of selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. A) A predetermined precursor ion (mass-to-charge ratio, m/z 521.7) is selected in the first mass analyzer (Q1), fragmented by collision-induced dissociation (CID), and one of the resultant product ions (m/z 757.6) is selectively monitored in the second mass analyzer (Q3). B) Example of four different peptides generated by protein digestion with the enzyme trypsin (i.e., tryptic peptides) measured by SRM in human plasma.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3860482&req=5

f10-arh-31-3-251: Illustration of selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. A) A predetermined precursor ion (mass-to-charge ratio, m/z 521.7) is selected in the first mass analyzer (Q1), fragmented by collision-induced dissociation (CID), and one of the resultant product ions (m/z 757.6) is selectively monitored in the second mass analyzer (Q3). B) Example of four different peptides generated by protein digestion with the enzyme trypsin (i.e., tryptic peptides) measured by SRM in human plasma.
Mentions: Using the selectivity of multiple stages of mass selection of a tandem mass spectrometer (see figure 10), these targeted SRM assays are the mass spectrometry equivalent of a Western blot.5 In fact, Arnott and colleagues (2002) originally coined the term “MS Westerns” for the use of tandem mass spectrometry to target individual hypothesized peptides. Just as a Western blot only produces a signal for proteins within a complex mixture that are recognized by an antibody, a targeted mass spectrometry assay will only produce a signal for peptides that have a specific combination of precursor and product ion m/z. This combination of precursor and product ion m/z is extremely selective and referred to as a SRM transition. The advantage of using a targeted mass spectrometry–based assay is that it does not require creating any immunoaffinity reagents.

Bottom Line: Proteins do not conform to any one uniform sample preparation method and/or biochemical analysis.Furthermore, because the number of biological replicates involved in behavioral analyses typically is high, robust high-throughput proteomic platforms will be required to handle the multitude of protein samples that can potentially result from the various brain regions for the numerous animal models and paradigms.Finally, these effects often are monitored over time courses, again inflating the total number of samples that need to be analyzed and compared.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences at the University of Washington, Seattle, Washington.

ABSTRACT
Alcohol addiction is a complex disease with both hereditary and environmental influences. Because molecular determinants contributing to this phenotype are difficult to study in humans, numerous rodent models and conditioning paradigms have provided powerful tools to study the molecular complexities underlying these behavioral phenotypes. In particular, specifically bred rodents (i.e., selected lines and inbred strains) that differ in voluntary alcohol drinking represent valuable tools to dissect the genetic components of alcoholism. However, because each model has distinct advantages, a combined comparison across datasets of different models for common changes in gene expression would provide more statistical power to detect reliable changes as opposed to the analysis of any one model. Indeed, meta-analyses of diverse gene expression datasets were recently performed to uncover genes related to the predisposition for a high alcohol intake. This large endeavor resulted in the identification of 3,800 unique genes that significantly and consistently changed between all included mouse lines and strains . Similar experiments also are crucial at the protein level. However, these analyses are not yet possible. Proteins do not conform to any one uniform sample preparation method and/or biochemical analysis. They display a broad range of physical and chemical properties (e.g., molecular weight or hydrophobicity) and are expressed over a very large dynamic range (up to 8 orders of magnitude). Further complicating global proteomic comparisons are the added considerations that proteins often undergo extensive covalent modifications and that protein functions often are regulated by complex protein-protein interactions and the specific location of the proteins in the cell (i.e., their subcellular localization)). Furthermore, because the number of biological replicates involved in behavioral analyses typically is high, robust high-throughput proteomic platforms will be required to handle the multitude of protein samples that can potentially result from the various brain regions for the numerous animal models and paradigms. Finally, these effects often are monitored over time courses, again inflating the total number of samples that need to be analyzed and compared. This article summarizes some general strategies for large-scale, high-throughput protein analyses and describes two new proteomic strategies that appear promising for future studies in this field.

Show MeSH
Related in: MedlinePlus