Protein quantification in ecological studies: A literature review and empirical comparisons of standard methodologies

Abstract

Protein quantification is a routine procedure in ecological studies despite the inherent limitations of well‐acknowledged protein determination methods which have been largely overlooked by ecologists. Thus, we want to bridge this knowledge gap, in hopes of improving the way ecologists quantify proteins and interpret findings. We surveyed the ecological literature to determine how and why ecologists quantify proteins. To determine whether different quantification methods produce comparable results across taxa, and between populations of a single species, we estimated the protein content of eight phylogenetically diverse taxa, and of desert isopods fed different diets, using various derived protocols of the 'crude protein', Bradford and bicinchoninic acid approach (BCA) methods. We found that ecologists use many protein quantification procedures, often without reporting the crucial information needed to evaluate and repeat their methods. Our empirical work demonstrated that the three quantification methods examined, and their derived protocols, resulted in highly divergent protein estimations that were inconsistent in rank across taxa, preventing conversion between methods. We also found that different quantification methods yielded different answers to whether isopod protein content is affected by diet. We conclude that commonly used quantification techniques yield distinct protein estimations with varying precision, and no single method is likely to be more accurate than another across taxa which may lead to inconsistent results across taxa and between conspecifics. Inaccurate protein quantification may explain the observed mismatch between organismal N and protein that has plagued some recent studies and that contradicts the principles of ecological stoichiometry. We recommend using a single BCA protocol to reduce inconsistencies across studies, until the promising amino acid analysis becomes more affordable, accurate and accessible to ecologists. Until then, ecologists should consider the abovementioned drawbacks of protein quantification methods and interpret their results accordingly.

Methodological limitations and conceptual implications of nutritional estimations

“…However, this explosion of quantification methods may harbor potential risks if used or reported erroneously, because many if not all of these methods are biased and sensitive to protocol changes. While these problems are wellknown in various fields such as biochemistry, pharmacology and nutrition, we found no similar discussion in the ecological literature except in the case of protein, recently reviewed by Zaguri et al (2021). Even more troubling is that ecologists seem to be largely oblivious to these problems, as reflected by the many nutrients estimation protocols used indiscriminately and the overwhelming tendency not to report crucial steps in the nutrients estimation protocols.…”

“…For proteins, we recommend using the BCA method (Smith et al 1985), following the suggestion of Zaguri et al (2021). BCA is fast, affordable and simple to master.…”

“…Even more troubling is that ecologists seem to be largely oblivious to these problems, as reflected by the many nutrients estimation protocols used indiscriminately and the overwhelming tendency not to report crucial steps in the nutrients estimation protocols. For example, only 61% of the 79 ecological studies that used the most prevalent Bradford method for protein estimation reported the type of standard used for calibration, only 8% reported the incubation time, and only 5% reported the incubation temperature that can all dramatically change the total protein estimation (Zaguri et al 2021). The lack of awareness is surprising given the potentially important empirical and conceptual implications of these methodological caveats for ecological research.…”

Protein sustained release from isobutyramide-grafted stellate mesoporous silica nanoparticles

“…Such difficulties come from the combination of several parameters: the amount of protein available for the assay (sensitivity), the composition of the sample (interferences), the amino-acid sequence of the protein (sensing specificity) and how tests are used and reported (operator). As an example, Zaguri et al identified 19 distinct methods to quantify proteins used by ecologists, and they notably noted the variability of how a given method can be used (difference in the incubation time, temperature…) and how the protocol can be reported ( Zaguri et al, 2021 ). To give a precise example, they found that only 8% of the studies using the Bradford assay in their corpus reported the incubation time, and only 5% reported the incubation temperature.…”

A Quick Analysis Method for Protein Quantification in Oilseed Crops: A Comparison With Standard Protocol

“…Additionally, the protein’s quantitative analysis is important for accurate labeling of food as well as its quality control ( 22 ). Primarily used methods for protein nitrogen determination in food samples has also been utilized for determining various other nitrogen forms in plant materials, soils, wastewater matrices, and biological tissues ( 23 , 24 ). To date, many methods have been developed for determining protein in food samples.…”