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Is Your Meat Tender? Proteomics May Provide the Answer


By Tony Fong

This story originally ran on June 24.

How tender is that cut of beef on your plate?

Up to now, there has been no truly accurate way of telling without biting into the meat, but using a mass spectrometry-based approach, researchers from Ohio State University performed a functional proteomics analysis that may eventually lead to a more precise method for predicting beef tenderness.

In a study published in the June issue of the Journal of Agricultural and Food Chemistry, the OSU researchers analyzed electrophoretic bands from the myofibrillar muscle fraction from 22 Angus cross steers and identified several protein trends that ultimately could be used to create a test to help cow farmers and US Department of Agriculture evaluators better set prices for beef based on tenderness.

Along with confirming earlier results by other researchers, the study presents new findings, forming the most complete picture yet of the mechanisms involved in meat tenderness, MacDonald Wick, an associate professor of animal sciences at OSU, and the corresponding author of the JAFC study, told ProteoMonitor this week.

"The community that studies this is in agreement that there are enzymatic processes that occur post-mortem over a period of several days that will give rise to a tender meat," Wick said, but "we don't know what those enzymatic processes are … and the only way that we believe that we can identify those enzymes [that] are involved in these post-mortem events is to identify the protein fragments themselves that are being generated."

While the authors wrote in the study that the comigration of proteins within an electrophoretic band prevents the definitive identification of proteins and peptides that play "a direct role in the mechanisms underlying post-mortem tenderness," Wick said that the work described in JAFC shows that "there are certain proteins or peptides or electrophoretic bands that we are absolutely positive are involved in the processes giving rise to tenderness."

Beef Down

The basis of the study is rooted in an effort to reverse a decline in beef consumption that began three decades ago as beef products lost market share to poultry. In an effort to understand this trend and to find ways of getting the public to eat more beef, a series of National Quality Beef Audits was conducted, first in 1991 and subsequently in 1995, 2000, and 2005. The audits found that beef quality, including tenderness, has been a major factor in the drop-off in consumption.

If the quality of the meat could be improved, consumers told pollsters conducting the audits, they would not only eat more beef but would be willing to pay more for it.

But determining beef tenderness, said Wick, is a tricky business. Beef carcasses are valued on the "potential palatability of the meat," Wick said, including its tenderness, juiciness, and flavor. These qualities are grouped into quality grades ranging from "Standard," the lowest, to "Prime," the highest.

The grades, though, are based essentially on the appearance and condition of the cow's carcass, and "the literature supports the contention that the current system is not very accurate," Wick said. "It is reported that there are too many carcasses with tender meat that are discounted and far too many with tough meat that are not discounted under the current USDA quality grading system."

In its study, the OSU team attributed the mechanisms involved in beef tenderness to a complex interplay of "cellular functions, which have proven difficult to develop into a coherent model," that include variations in genetics, final pH, the proteosome, and heat shock proteins.

Functional proteomics, they said, offers a novel approach at learning more about the mechanisms that control meat tenderness.

The purpose of the study "was to perform a functional proteomics analysis to associate electrophoretic bands from the myofibrillar fraction of meat samples at 36 hours post-mortem that are statistically significant with meat tenderness at 72 hours and 14 days of aging and/or the tenderness differential and determine the sequence of the protein(s)/peptide(s) in those bands," they wrote.

In short, their research comprised an analysis by SDS-PAGE of myofibrillar muscle fractions from Angus breeds. The fractions were then linearly regressed to values of a common method for assessing beef tenderness, Warner-Bratzler shear. Significant electrophoretic bands, six out of a total of 30, were then characterized by electrophoretic and statistical analysis and sequenced by nano-LC-MS/MS.

Within the six bands a total of 36 proteins and peptides were identified and determined to be associated with beef tenderness, though some of them were identified in more than one band. Included among those that appear to be participating in meat tenderness are proteins/peptides that have structural, metabolic, chaperone, and developmental functions.

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In their mass spec work, the researchers reported encountering comigration of proteins and peptides within a given band — a band may contain many spots, each containing more than one protein — which Wick and his team navigated around by choosing "those proteins [that] make sense to us in terms of our understanding of the biochemistry and the current literature."

Some technologies, such as iTRAQ, would have allowed them to avoid this problem, Wick said, "but for us to look at trying to associate changes in the proteome with the changes in an independent variable in which you use 70 animals, for instance," would be too cost- and labor-prohibitive.

To solve this, the researchers are currently developing an algorithm that would "analyze the mass scans to determine the changes in the concentrations of each peptide in order to determine the contributions of each of the comigrating peptides to the differential band intensities over the range of the independent variable, such as tenderness values or growth rate," Wick said.

They also are working on new mass-spec strategies such as using longer columns for improved resolution, he added.

A Beef Narrative

According to Wick, one of the values of the study is that it begins to a more complete story about the role proteins may play in beef tenderness by linking the findings of earlier work along with these new findings: If earlier studies are sentences, the JAFC study links the sentences together into a paragraph that begins to form a narrative.

"What we have been able to do is grab [all the individual studies] at one time and show that they are actually part of the same coherent story, which has not been done before," Wick said.

Indeed, he and his co-authors present several trends that they said could explain meat tenderness. One trend of particular interest to Wick is the presence of myosin proteins, structural proteins that are specific to the sarcomere, the smallest functional unit of the myofibril. Myosin heavy chains and myosin light chains "tended to be found in bands associated with all [Warner-Bratzler shear] variables," the authors wrote in the study.

Myosin heavy chain degradation during post-mortem events has not been a widely accepted explanation for meat tenderness, but "people are [coming] on board with that," and the study further supports the theory, Wick said. Why that process occurs and how it works is still not understood, however.

One trend that the authors said is unique to their study related beef tenderness to muscle differentiation. The researchers discovered a protein, cysteine-rich and glycine-rich protein 3, also known as muscle LIM, which acts like a zinc finger. According to the study, the RNA expression of the protein "has been observed to be increased in muscle tissue of beef cattle undergoing nutritional stress." The OSU study, however, is the first linking the protein to tenderness, the authors said.

"What we think we found here [is] this LIM is somehow — and we have no idea what the heck is going on here either — participating in changing the ultimate fiber type of [the] animal, which is then related to tenderness," Wick said.

Another area they "absolutely" investigate is the role that post-translation modifications may play. In the present study, which was a 1D study, the idea was to "find a cadre of proteins" implicated in meat tenderness. Now they can take two pieces of beef, one tough, one tender, and do a DIGE experiment. If the same proteins are found in the two samples, "by golly those are post-translationally modified. You're pretty sure they are, especially if they're around the same molecular weight," Wick said.

So, if the OSU researchers' findings pan out and a proteomics workflow can be used to predict which steak will melt in your mouth, should we expect every Piggly Wiggly market and Smith & Wollensky steakhouse to be equipped with a mass spec so they can determine the tenderness of their cuts and price them accordingly?


What Wick foresees is the development of a test strip, similar to those used for home pregnancy tests, that could be used during meat processing for the quantitative detection of proteins that are predictive of tenderness.

"A system based on the biology of post-mortem meat would be more accurate" than the existing methods for determining meat tenderness, Wick said. "We believe that we could do that using one or more of the proteins identified in our studies as antigens for the development of a rapid immunochemical-based assay for use in the processing facility."

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