Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization

Abstract

Post translational modification (PTM) and proteolytic processing of proteins contributes to regulation of their stability, intracellular localization and interactions with other proteins, and to direct enhancement or repression of their activity. Both PTM and proteolysis are dynamic; levels and rates change in response to changes in the cellular environment. Tissue excision, post mortem interval and subsequent methods of tissue processing for protein analysis unavoidably alter the cellular environment and therefore features of protein profiles. To aid in understanding the time frame and protein specificity of these changes and the biological and technical contributions to them, we have compared features of protein profiles in mouse hippocampus and cortex following three methods of tissue handling: immediate lysate preparation, and rapid heating to 95°C and standard snap freezing in liquid nitrogen, prior to lysate preparation. In spite of the very short time frames involved, we observe protein-specific differences in levels of phosphorylation, general differences in patterns of sumoylation, and specific differences in levels of proteolytic cleavage of calcineurin and the neurotrophin receptor, TRKA. These differences vary with brain region and with post excision time to processing, and highlight the challenges inherent in accurately profiling the in vivo proteome.

Figure 1. Patterns of phospho-serine appear similar in frozen and heat stabilized cortex

Lysates were prepared after snap freezing in liquid nitrogen (FZ1-FZ3) or heat stabilization (ST1-ST3). Immediately prior to lysate preparation, brain regions were cut in half; one half was immediately placed in IEF buffer and processed (0 minutes room temperature (0 min RT)) and the other half was held for 30 minutes at room temperature (30 min RT) followed by buffer addition and processing. Gel membrane was screened with an antibody to phospho-serine.

Figure 2. Hippocampal levels of specific phospho-proteins differ between frozen and heat stabilized tissues and between 0 and 30 minutes room temperature incubation

Lysates were prepared from hippocampus from the same frozen (FZ1-FZ3) and stabilized (ST1-ST3) brains as in Figure 1. Gel membranes were screened with antibodies to the indicated proteins.

Figure 3. Quantitation of phospho-proteins in hippocampal lysates shows that heat stabilized levels remain unchanged while frozen tissue levels are decreased with room temperature incubation

Signals from replicate Western blots as in Figure 2 were normalized to actin. Histograms represent average values for each group of samples relative to 100% for frozen tissues at 0 min RT (white bars); black bars, stabilized tissues 0′ RT; grey bars, frozen tissues 30′ RT; striped bars, stabilized tissues 30′ RT. Errors bars are the standard error of the mean. Statistical significance was determined by the unpaired Student's t-test. *p<0.05; **p <0.001:***p <0.0001.

Figure 4. Cortex levels of specific phospho-proteins differ between frozen and heat stabilized tissues and between 0 and 30 minutes room temperature incubation

Lysates are those used in Figure 1. Gel membranes were screened with antibodies to the indicated proteins as in Figure 2.

Figure 5. Unchanged levels of phospho-proteins in cortical lysates from heat stabilized tissue and decreased levels in frozen tissue with room temperature incubation

Signals from replicate Western blots as in Figure 4 were normalized to actin. Histograms represent average values for each group of samples relative to 100% for frozen tissues at 0 min RT (white bars); black bars, stabilized tissues 0′ RT; grey bars, frozen tissues 30′ RT; striped bars, stabilized tissues 30′ RT. Errors bars are the standard error of the mean. Statistical significance was determined by the unpaired Student's t-test. *p<0.05; **p <0.001:***p <0.0001.

Figure 6. Decreased levels of sumoylation and increased proteolytic processing of calcineurin and TRKA in lysates from frozen vs. stabilized cortex

Samples are the same as in Figure 4. (a) Free SUMO3 monomers, dimers and trimers are indicated. Higher molecular weight bands are SUMO-modified proteins. A faint smear between 72 and 95 kD is visible on the frozen (FZ) samples. (b) Calcineurin catalytic subunit, CaNA major signal at ∼60 is indicated. A smaller fragment at ∼30 kD is seen only in FZ samples. (c) TRKA signal is indicated at ∼140 kD; a TRKA-specific fragment at ∼35 kD is seen only in FZ samples.