PubMed · 14197017
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M K SEIKEL, M A MILLETT, J F SAEMAN. 1964. NOTES ON SEMIMICRO PREPARATIVE THIN-LAYER CHROMATOGRAPHY.. https://doi.org/10.1016/s0021-9673(01)82758-8
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The whole proteome of any organism is too complicated to be analyzed in a simple one-step process and direct attempts for the entire proteome analysis normally lead to considerable loss of information. A practical approach is the targeting of the specific structural feature of interest using chromatography. This approach simplifies the proteome while preserving most of the vital information necessary for analysis. Selection of peptides with specific amino acids (cysteine, histidine and methionine) or N- or C-terminal peptides is an accepted procedure for proteome simplification when general analysis is desired. While selection of enzymatically and non-enzymatically modified proteins and peptides is used when post-translational modifications are targeted. Protein interaction with small molecules as well as other proteins also has been studied using chromatographic selection methods.
This article attempts to answer the question of how many replicate sample preparations and replicate chromatographic injections must be done to provide accurate results in chromatographic analyses of pharmaceuticals. Using a random selection of chromatographic runs obtained with 1-3 replicate preparations and duplicate injections, the variance associated with preparation-to-preparation and injection-to-injection variability were estimated by a mixed-model statistical analysis. The analysis also predicted the probability that two injections of the same sample preparation are not in agreement with each other. Results indicated that, with modern chromatographic equipment, duplicate injections do not improve the precision. The number of replicate preparations needed to provide accurate results for various types of analysis depends on the type of sample and the desired tightness of the specification limits.