Analytical and biological variances associated with proteomic studies of Medicago truncatula by two-dimensional polyacrylamide gel electrophoresis

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry are being used as proteomic tools in an integrated functional genomics program focused on the model legume Medicago truncatula. Due to the perceived high levels of indeterminate error associated with 2-DE we deemed it necessary to quantify the coefficient of variance (or relative standard deviation) for both analytical and biological sources associated with 2-DE of Medicago truncatula leaf protein extracts. Leaf protein extracts were chosen because of their biological significance and due to the more challenging nature of green tissues. Analytical variance was calculated for fifty proteins from ten replicate 2-DE gels of the same protein extract. Biological variance was calculated for the same fifty proteins from ten independent 2-DE gel analyses of ten independent but similar plants grown under identical conditions. Average analytical and biological variances were calculated for both data sets and represent the average variance of approximately 500 independent measurements of protein concentration. Analytical variance was determined to be 16.2% and biological variance was determined to be 24.2%. These average variances provide a quantified and statistical basis for evaluation of protein expression changes in future comparative proteomic investigations. It is proposed that 2-DE measured protein expression levels should differ by a minimum of 3.92sigma (i.e. /+/-2sigma/ and sigma = standard deviation), or 94.7% based on our measured variances, for the difference to be significant at the 95% confidence level.

Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance

“…The experiments were set up in randomized block design where six gels corresponding to independent protein extractions from various desiccationtolerant and -sensitive stages were run in parallel. Four to eight gels per stage were accumulated independently until the coefficient of variance of the normalized intensity of 50 representative spots that were present in all stages was below 25% (Asirvatham et al, 2002).…”

A Two-dimensional Electrophoresis Proteomic Reference Map and Systematic Identification of 1367 Proteins from a Cell Suspension Culture of the Model Legume Medicago truncatula

“…relative standard deviation) for the total number of protein spots detected was determined to be 6%, and the variance in normalized spot volume was determined to be 48%. These values are similar to those reported by others in the literature for 2-DE (47,48).…”

Variability of healthy human proteome

“…К настоящему времени описана вариабельность белков тканей различных организмов. Например, коэффициент вариации белкового состава растительных тканей составил 24% (при ошибке метода 16%) [40], а животных (грызунов, свиней) -от 40 до 60% (при ошибке метода 15-23%) [41,42]. По данным Molloy с соавт., для клеточных линий бактерий и млекопитающих вариабельность равна 24-47% [43].…”