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University of Texas MD Anderson Cancer Center

Comparison of Pinnacle and SameSpots for Spot Quantification and Differential Expression in 2-Dimensional Gel Electrophoresis Studies
Jeffrey S. Morris Sun, 15 Feb 2009 04:27:12 -0800
2-DE is an important method for proteomics. Accurate spot detection and quantification on the resulting images is a challenging task, but must be done effectively for the technology to fulfill its potential. Traditional analytical methods have significant weaknesses, including spot mismatching and missing data. These problems require time-consuming manual editing to correct, which dramatically decreases throughput and compromises the objectivity and reproducibility of the analysis. To address this issue, we developed Pinnacle, a new method that markedly improves spot detection and quantification precision. Another new method implemented in Progenesis SameSpots, a commercial 2-DE analysis product, has also been touted as an improvement upon traditional approaches. In this study, we compared Pinnacle and SameSpots in spot detection and precision of quantification using two different dilution series, and evaluated the detection of differentially expressed proteins in two differential expression experiments. We found that SameSpots at times had problems with spot delineation, while Pinnacle did not. While both Pinnacle and SameSpots showed marked improvement in precision of spot quantifications over conventional methods, Pinnacle yielded spot quantifications with greater validity and reliability than SameSpots. Pinnacle detected more differentially expressed proteins than SameSpots, which may be a result of its increased precision and improved spot delineation.
Statistical Issues in Proteomic Research
Jeffrey S. Morris Thu, 31 Jul 2008 11:03:24 -0700

Microproteomics: Analysis of protein diversity in small samples
Howard B. Gutstein Fri, 13 Jun 2008 14:38:53 -0700
Proteomics, the large-scale study of protein expression in organisms, offers the potential to evaluate global changes in protein expression and their post-translational modifications that take place in response to normal or pathological stimuli. One challenge has been the requirement for substantial amounts of tissue in order to perform comprehensive proteomic characterization. In heterogeneous tissues, such as brain, this has limited the application of proteomic methodologies. Efforts to adapt standard methods of tissue sampling, protein extraction, arraying, and identification are reviewed, with an emphasis on those appropriate to smaller samples ranging in size from several microliters down to single cells. The effects of miniaturization on these analyses are highlighted using neuroscience-related examples, as are statistical issues unique to the high-dimensional datasets generated by proteomic experiments.
Pinnacle: A Fast, Automatic Method for Detecting and Quantifying Protein Spots in 2-Dimensional Gel Electrophoresis Data
Jeffrey S. Morris Tue, 04 Dec 2007 09:44:53 -0800
Motivation: One of the key limitations for proteomic studies using 2-dimensional gel electrophoresis (2DE) is the lack of rapid, robust, and reproducible methods for detecting, matching, and quantifying protein spots. The most commonly used approaches involve first detecting spots and drawing spot boundaries on individual gels, then matching spots across gels, and finally quantifying each spot by calculating normalized spot volumes. This approach is time con-suming, error-prone, and frequently requires extensive manual edit-ing, which can unintentionally introduce bias into the results.Results: We introduce a new method for spot detection and quanti-fication called Pinnacle that is automatic, quick, sensitive and spe-cific, and yields spot quantifications that are reliable and precise. This method incorporates a spot definition that is based on simple, straightforward criteria rather than complex arbitrary definitions, and results in no missing data. Using dilution series for validation, we demonstrate Pinnacle outperformed two well-established 2DE analysis packages, proving to be more accurate and yielding smaller CVs. More accurate quantifications may lead to increased power for detecting differentially expressed spots, an idea supported by the results of our group comparison experiment. Our fast, automatic analysis method makes it feasible to conduct very large 2DE-based proteomic studies that are adequately powered to find important protein expression differences.Availability: Matlab code to implement Pinnacle is available from the authors upon request for non-commercial use.
Laser capture sampling and analytical issues in proteomics
Howard Gutstein Tue, 04 Dec 2007 09:35:54 -0800
Proteomics holds the promise of evaluating global changes in protein expression and post-translational modificaiton in response to environmental stimuli. However, difficulties in achieving cellular anatomic resolution and extracting specific types of proteins from cells have limited the efficacy of these techniques. Laser capture microdissection has provided a solution to the problem of anatomical resolution in tissues. New extraction methodologies have expanded the range of proteins identified in subsequent analyses. This review will examine the application of laser capture microdissection to proteomic tissue sampling, and subsequent extraction of these samples for differential expression analysis. Statistical and other quantitative issues important for the analysis of the highly complex datasets generated are also reviewed.
Statistical contributions to proteomic research
Jeffrey S. Morris Wed, 04 Apr 2007 12:55:09 -0700
Proteomic profiling has the potential to impact the diagnosis, prognosis, and treatment of various diseases. A number of different proteomic technologies are available that allow us to look at many proteins at once, and all of them yield complex data that raise significant quantitative challenges. Inadequate attention to these quantitative issues can prevent these studies from achieving their desired goals, and can even lead to invalid results. In this chapter, we describe various ways the involvement of statisticians or other quantitative scientists in the study team can contribute to the success of proteomic research, and we outline some of the key statistical principles that should guide the experimental design and analysis of such studies.

 

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