Sample preparation - a necessary evil or the key to proteomics?

Mats Borén, Head of Development, Denator.

Over the years, the main focus of proteomics has been on technological aspects such as capacity and dynamic range of 2D gels, masspectrometry resolution and sensitivity. Enormous amounts of money and effort have been spent on this work while the softer side of proteomics; study design, sample preparation and reproducibility have been left out in the cold. The old saying "garbage in, garbage out" holds as true for proteomics as other parts of life, no matter how many millions of Euros you spend on for instance mass spectrometers. In order to fix proteomics and release its full inherent potential these softer aspects must be allowed a place on centre stage.

A provocative paper was published last year by Petrak et al. showing that a small number of proteins significantly changes in study after study regardless of disease state in both human and rodent 2DE based experiments [1]. This, rather shocking, insight raises questions of systematic technical artefacts, limitations or bias of the methods utilized. Recent studies of post-mortem changes in tissue have identified a number of proteins that appear extra sensitive to post-mortem degradation [2]. Several of those proteins correspond with those identified in the Petrak study. Coincidence? What is the cause and what is the effect? Inflammation has been suggested as a possible explanation but it could just as well be due to current sampling practise and post-sampling protein changes and degradation. Millions of Euros are spent each year on chemical enzyme inhibitors that are added, rather indiscriminate, to samples with the intention of preventing post-sampling changes. Have you ever tested if they work or are you just satisfied with doing what everybody else is doing?

Taking a sample is a major traumatic event with drastic effects on the cellular level even within a very short timeframe. It is important to remember that although the sample has been removed from its source, it is still very much alive and cellular activity continues and tries to adapt to the new situation. Upon sampling, oxygen levels drop dramatically leading to energy depletion and membrane potential breakdown, which cause the carefully orchestrated cellular machinery run amok.

Figure 1 The number of detected peptides in mouse hypothalamus increase with post-mortem time. Two-dimensional graphs of samples completely inactivated at 0 min and 10 min post-mortem, respectively [2].

Rapid freezing of the sample temporarily pause this activity but also induce further rupturing of membranes and destruction of cellular structures unless special measures are taken [3]. Upon thawing, normally spatially restricted proteolytic enzymes come into contact with unintended targets, resulting in massive changes to protein integrity and modifications. The addition of inhibitors gives only partial alleviation since the inhibition effect is incomplete and the molecules take their time to reach intended targets. The use of denaturant solvents for extraction is not a sure way of avoiding protein degradation since evidence of proteolytic activity has been proven in both urea-based IEF buffer [4] and mixed organic-aqueous solvents [5].

Figure 2 Phosphatase activity measured using SensoLyte pNPP phosphatise kit (Anaspec). Mouse brain cortexes were snap frozen and samples were solubilised using sonification in 1X PBS, pH 7.4 from Anaspec with or without chemical inhibitors. Activity was reduced in samples where inhibitors had been added but not eliminated. [3]

The use of thermal inactivation as a means of eliminating enzymatic activity in biological samples has been reinvented. Thermal treatment, under standardized conditions, can be done directly after sample extraction, while enzymes still are within their correct environment, resulting in the stabilization of protein composition at the time of treatment [3]. This has obvious advantages over alternative approaches which are introduced at later stages in the workflow when degradation has already started taking place and that only reduces activity, not eliminating it. As an added benefit, no additives to the sample are required, interfere in subsequent steps is thus avoided.

The importance of correct sample preparation can not be overstated. Careful planning and meticulous attention to details in set-up procedures must be followed in order to get successful, reproducible and biologically meaningful results from proteomics studies. The mainstream practices of inhibitors and freezing are not satisfactory and the introduction of thermal stabilization appears as an interesting alternative.

Figure 3 Overview of the rapid effect of post-mortem degradation and changes throughout sample preparation.

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Sample preparation - a necessary evil or the key to proteomics?

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