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1885Bioanalysis (2015) 7(15), 1885–1899 ISSN 1757-6180

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Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (≈1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

BackgroundRecent advances of MS-based techniques for proteomics, peptidomics, metabolomics and tissue imaging have allowed researchers to proceed from identification and localiza-tion to quantification of target analytes [1–6]. There is increasing evidence that a majority of all proteins are expressed in all cell types and tissues in the human body and that tis-sue-specific cell properties are achieved by precise regulation of levels of proteins rather than which proteins are produced [7]. There is a need, not the least in clinical research and diagnostics, for analytical procedures which provide data with sufficient accuracy, precision and reproducibility [8,9] and which also, in the case of proteins, are selective for post-translational modifications. Therefore, sampling protocols that minimize altera-tion of sample components during and after sampling, prior to sample analysis are of the utmost importance. At the same time, biosa-mples collected from studies involving infec-tious and pathogenic microorganisms must face an additional challenge of pathogen inactivation for safe transport, and before downstream analysis, for example, studies on the proteome or determination of thera-

peutic drugs in tissue samples, can be safely performed in a routine laboratory environ-ment (biosafety level 2). Sample composition should as far as possible reflect the state of the living organism at the moment just before the sample is taken. Blood and tissue samples taken from living organisms will inevitably undergo changes in conjunction with and after sample collection. While blood sam-pling is relatively rapid, tissue sampling is more complicated and may involve a con-siderable time period of physiological stress and ischemia. After tissue excision, further microscope dissection may be needed prior to downstream homogenization and analy-sis [10,11]. For both sample types, there are analytical targets undergoing rapid conver-sion making adequate sampling a challenge. The scope of sample analysis may either be narrow, aiming at specific predefined tar-get analytes, or broad, covering a diversity of known and unknown compounds. For tissue samples, the spatial distribution of sample constituents is of interest and may be affected by the sample handling procedure. In targeted analysis, post-sampling changes may be acceptable as long as they do not affect the measurement of the analytes of

Thermal inactivation of enzymes and pathogens in biosamples for MS analysis

Martin Ahnoff*,1,2, Lisa H Cazares3,4 & Karl Sköld2,5

1Department of Chemistry & Molecular

Biology, University of Gothenburg,

SE-412 96 Gothenburg, Sweden 2Denator AB, Göteborg, Sweden 3Molecular & Translational Sciences,

United States Army Medical Research

Institute of Infectious Disease, 1425 Porter

St. Frederick, MD 21702, USA 4DoD Biotechnology High Performance

Computing Software Applications

Institute, Telemedicine & Advanced

Technology Research Center, US Army

Medical Research and Materiel Command,

Fort Detrick, MD 21702, USA 5Department of Medical Sciences, Cancer

Pharmacology & Computational Medicine,

University of Uppsala, Uppsala, Sweden

*Author for correspondence:

Tel.: +46 705191514

[email protected]

For reprint orders, please contact [email protected]

1886 Bioanalysis (2015) 7(15) future science group

Perspective Ahnoff, Cazares & Sköld

interest. Sample quality in this case can be reduced to requirements of stability of the analytes in the sam-ple matrix, and to measurement of these analytes not being affected by changes in the sample matrix [12]. With a broader analytical scope, the aim is to more generally inhibit processes that potentially alter sample composition, including, primarily, enzyme-catalyzed reactions. Enzymes are present in the interior of cells, on cell membranes (ecto-enzymes) and outside cells (exo-enzymes), for example, in interstitial liquid and in blood plasma. Injured or lysed cells will expel enzymes, which may promote enzymatic degradation, for exam-ple, after a freeze-thaw cycle. Enzyme-catalyzed pro-cesses occur rapidly, making sampling a real challenge. Cell signaling systems are important examples of such

processes. Protein phosphorylation at Tyr and Ser/Thr residues controls the activity of signal transduction pathways regulated by kinases and phosphatases [13,14]. Lysine acetylation has been less studied than phos-phorylation but is estimated to have a broad range of functions in cell signaling. Acetylation of lysine resi-dues in the active site of a protein affects its activity and can interact with protein phosphorylation [15,16].

Chemical biomarkers of disease and disease state may be macromolecules (proteins or larger peptides) or small molecules (lipids [11], glycans, metabolites). In the search for biomarkers, a criterion should be that they are not subject to change in the pre-analytical phase [17,18]. Preanalytical degradation limits the dis-covery of potential biomarkers. In addition, consider-ing that the combination of a panel of biomolecules rather than a single component may serve as a bio-marker, rapid enzyme inactivation procedures will effectively broaden the range of potential biomarkers discovered in any given experimental procedure.

Tissue proteomic and metabolomic investigations are increasingly employed for biomarker discovery and disease mechanism investigation. Examination of pathogen infected tissue enables the elucidation of host-pathogen interactions leading to a better biologi-cal understanding of disease pathogenesis, and allow-ing researchers to zone in on potential targets for anti-microbial therapeutics as well as promising antigens for vaccine development [19]. Therefore, the choice of methods to eliminate or inactivate a microbial biohaz-ard for the safe laboratory handling, while stabilizing the tissue to preserve molecules of interest is critical to the advancement of infectious disease research. A variety of inactivation treatment techniques are avail-able, but practicality and effectiveness often govern which is most appropriate, especially when handling tissue in biocontainment laboratories. The ideal tissue fixation method for downstream analysis of proteins, peptides and metabolites would inactivate pathogens, while preserving the molecules of interest in their ‘in situ’ state.

Biosample preservationTissue sample fixation & stabilizationTissue samples taken for histology, pathology or cell biology investigations are preserved from decay by fixation, preventing autolysis and putrefaction. Among the different chemical treatments used, treatment with 10% neutral buffered formalin (NBF, containing ~3.7–4.0% formaldehyde and 1% methanol in phos-phate buffered saline) has been widely used as a stan-dard procedure. Formaldehyde reacts primarily with free amino groups (e.g., lysine) and thiols (cysteine). Consecutive reactions with primary amides (glutamine,

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Key terms

Pathogen inactivation: Process designed to kill or destroy disease-causing agents such as bacteria and viruses.

Enzyme inactivation: Process designed to change the state of proteins, resulting in loss of their properties as catalysts of biochemical reactions.

Tissue fixation: Step in the preparation of histological sections, by which biological tissues are preserved from decay, preventing autolysis or putrefaction. Normally involves treatment with chemicals.

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Figure 1. Degradation of fibrinogen peptide A peptide in plasma samples. Plasma was obtained using blood collection tubes containing heparin, citrate or EDTA, or EDTA plus proprietary protease inhibitors (P100) and was spiked with stable-isotope labeled fibrinogen peptide A (FPA) peptide, and quantified after different times of incubation using MALDI–MS. The peak intensities of the FPA peptide in natural logarithm are plotted versus incubation time. For all except P100 samples, peak intensities were at or below the detection limit at the last timepoint shown. EDTA: Ethylenediaminetetraacetic acid. Reprinted with permission from [25] © American Chemical Society (2008).

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Thermal inactivation of enzymes & pathogens in biosamples for MS analysis Perspective

asparagine), guanidine groups (arginine) and tyrosine ring carbons results in cross-linking. Formaldehyde also cross-links RNA with proteins [20] and reacts with small molecules, for example, benzodiazepines [21]. At room temperature, the fixation process takes approxi-mately 24 h to be complete, although penetration of a tissue sample, depending on sample thickness, can be much faster. Despite the many advantages of for-malin fixation, there are also several drawbacks [22], especially when the samples are to be used for molecu-lar diagnostics. For that reason, an alternative to for-malin fixation for chemical fixation and stabilization was developed [23] and reagents were commercialized under the trademark PAXgene. Fixation, using a mix-ture of different alcohols, acid and a soluble organic compound, is followed by stabilization using a mix-ture of alcohols, to be added to the fixed sample. This fixation process is not dependent on slow cross-linking reactions as is the case with formalin fixation, but is still dependent on the time needed for the reagents to penetrate through the tissue. Recommended fixa-tion time is 2–4 h according to the manufacturer. In order to preserve biomolecules, which are significantly affected by processes within this timeframe, there is a need for faster stabilization of tissue samples than what can be achieved by chemical reagents. Furthermore, small molecules such as amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), have been shown to leach from tissue sections into the aqueous fixative medium, creating a source of variability in molecular content [24].

Protein denaturation & enzyme inhibitionEnzyme inhibitors are often used with the aim to pre-serve proteins in their native state. When an inhibi-tor acts through competitive reversible binding, this requirement may be fulfilled, but such inhibition is often not total. For example, Yi et al. [25] showed that the sta-bility of fibrinogen peptide A in plasma using different anticoagulants increased in the order heparin < citrate < EDTA. Stability was further enhanced using P100 blood collection tubes, containing K

2EDTA and pro-

prietary protein stabilizers, so that a half-life of 12.3 h was achieved while for another peptide (SSKITHRI-HWESASLLR) a half-life of only 0.34 h was observed (Figure 1). Some inhibitors, such as phenylmethanesul-phonyl fluoride and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, bind irreversibly to the active site of an enzyme, which means that these proteins have been modified and are no longer in their native state [26]. EDTA inhibits reactions by chelating metal ions and has been shown to cause irreversible denatur-ation and aggregation of zinc-binding proteins. Cool-ing is widely used for temporary inhibition of enzyme

activity. Many enzymes in endothermic organisms lose their activity at temperatures near zero degrees Celsius. Such cold denaturation is most often reversible. Quick deep-freezing to -70°C or colder will effectively stop all enzyme activity, but after thawing, enzyme activity is largely restored (or increased due to cell lysis) while the freeze/thaw cycle also may result in irreversible partial denaturation of proteins [27]. To summarize, effective inhibition of enzyme activity cannot be done without at least some irreversible modification of proteins, and it can be argued that the only practical way to stop all enzyme activity completely is to denature all the proteins in a sample.

The term denaturation denotes changes or altera-tions of the secondary and higher structures of a pro-tein molecule resulting in loss of its specific function. The protein is transformed from its ordered, native, state to a less-ordered state due to rearrangement of hydrogen bonding [27]. As described by Murphy [28] the dominating interactions determining the stability of a protein are the hydrophobic effect (low-relative solu-bility of apolar groups), hydrogen bonding (competi-tion between bonding within the folded protein and bonding to solvent water) and configurational entropy (loss of configurational entropy for the highly ordered folded protein compared with the less ordered unfolded

1888 Bioanalysis (2015) 7(15)

Figure 2. Ex vivo protease activity in snap frozen or heat stabilized rat brain tissue samples studied by oxygen-18 incorporation and nano-flow LC–MS/MS. Peptides were extracted using 8 M urea and stored under different conditions (urea concentration, temperature and time) in the presence of 50% H2

18O, simulating conditions typical for various analytical scenarios: peptide extraction workflow (pep and low urea), shotgun sample preparation (shotgun), isoelectric focusing stage of 2D-gel electrophoresis sample preparation. Filled squares in the plot show proportion of total number of identified peptides with oxygen-18 incorporated at the C-terminal, in individual samples (n = 4) from each treatment. The graph shows that samples, after treatment with strong denaturing buffer, were to a varying degree exposed to protease activity. Initial heat stabilization resulted in low protease activity in contrast to initial snap-freezing. D: Heat stabilized; SF: Snap frozen. Reprinted with permission from [30] © American Chemical Society (2014).

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protein). Calculations of contributions to Gibb’s free energy from hydrophobic effects and hydrophilic effects (hydrogen bonding) and their temperature dependence are difficult, and the relative importance of hydrophobic and hydrophilic effects has recently been discussed [29]. Stability of a protein’s native state is dependent on its micro-environment (the solvent, other solutes, pH) and on temperature, each protein having its temperature window where its active conformation is stable. This window can be narrowed, or widened, by changing the chemical environment. Additives that weaken the native state (chaotropic agents), which are used for denaturation include alcohols, detergents, urea, thiourea and guanidine hydrochloride. Except for the alcohols, these agents tend to solubilize the pro-tein at concentrations needed for denaturation. High concentrations (≥8 mol/l) of urea result in strong dena-turation of proteins, while at lower urea concentration

(4 mol/l), enzymatic degradation of proteins has been observed in brain tissue samples (Figure 2) [30].

Procedures aiming at general denaturation of pro-teins in tissue samples can be employed as long as they are not in conflict with the aim of analysis and com-patible with analytical procedures. Achieving dena-turation without the use of chemical additives would be advantageous. Once added, they may be difficult to remove from the sample and could interfere with sample analysis. Unless the biosample is a semiliquid (blood, tissue homogenate) or a very thin tissue slice, penetration of the denaturing agent takes consider-able time during which inner parts of the sample are left unprotected. Proteins can be denatured quickly by increasing temperature beyond the temperature window where the protein folding is stable. Such heat denaturation typically is irreversible. While some (mammalian) proteins lose their stability above 38°C, others maintain their native state and activity at higher temperatures, and may require heating to 50–80°C for complete denaturation. Proteins in organisms liv-ing and thriving at high temperatures (thermophiles) are stable at such temperatures, which in extreme cases may be higher than 100°C. But there is no evidence of proteins in mammals and other vertebrates that are not denatured when heated to 85°C in their normal physiological environment.

Denaturation by heat is studied by differential scan-ning calorimetry [31]. The native and denatured states are thought to be in equilibrium as long as the dena-tured protein is not involved in secondary, irreversible, processes. In reality, denaturation often is accompa-nied by secondary reactions between the denatured protein molecule and its surroundings, including other protein molecules, which can lead to aggregation, coagulation, gelation and adhesion to surfaces. While the primary step involves heat uptake, in other words, is endothermic, the secondary processes typically are exothermic. The influence of secondary interactions can be decreased by using dilute protein solutions when studying heat denaturation by differential scanning calorimetry (DSC). Figure 3A & B shows thermograms of proteins which are major constituents of plasma, at concentrations corresponding to a 25-fold dilution. A temperature of around 90°C is sufficient for denatur-ation of all proteins. Heat denaturation of proteins in tissue samples, whole blood and plasma is irreversible, as can be shown by a second DSC temperature scan (Figure 3C).

Removing much of the water in a sample increases temperature stability of proteins, so that higher temper-atures are needed for denaturation (Figure 3D) [32]. This means that when the aim is to denature the proteins, drying (including lyophilization) of the sample prior


Figure 3. Heat denaturation of proteins studied by DSC and dependency of denaturation temperature on moisture content. (A) Calculated thermogram (dashed line) obtained from the sum of weighted contributions of the 16 most abundant plasma proteins (solid lines). (B) Thermograms obtained from mixtures of pure plasma proteins, mixed at concentrations that mimic their known average concentrations in normal plasma. Scans were recorded from 20 to 110°C at 1°C/min (MicroCal, MA, USA). The red curve is a mixture of HSA, IgG, fibrinogen and transferrin. The black curve is a mixture of the 16 most abundant plasma proteins. (C) Rat tail collagen protein denaturation measured by DSC. Solid line (i) shows heat uptake during heating from 20 to 90°C at 10°C/min. Dashed curve (ii) is from a second identical scan after returning to room temperature. (D) The dependence of the temperature of denaturation of egg albumin upon the relative humidity (%). Δ experimental heating time 10 min, O experimental heating time 60 min. (A & B) Reprinted with permission from [62] © Elsevier (2008). (C) Reprinted with permission from [27] © Wiley & Sons (2006). (D) Reprinted with permission from [32] © Rockefeller University Press (1933). For color images please see online www.future-science.com/doi/full/10.4155/bio.15.122

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to heat denaturation should be avoided. Considering that the optimal temperatures for enzymes in mam-mals typically are close to normal body temperature, 36–40°C, heat denaturation should be rapid, espe-cially over the temperature range of about 20–60°C, to avoid enzyme-driven reactions taking place during the heating-up phase, which could otherwise promote, rather than inhibit, degradation processes in the sam-ple. Heating should also be precise, bringing all parts of the sample to a temperature within a narrow range, which should be above, for example, 85°C to ensure complete denaturation and below the boiling point of water (100°C) to avoid damage of tissue structure and unwanted loss of water.

Both microwave irradiation [33] and conductive heating [34] has been used for heat denaturation of proteins in tissue samples. While conductive heating can be carried out on excised tissue samples, up to 7 mm thick, with high accuracy and precision [35], microwave irradiation can be used for in situ treat-ment and has a role in special applications, such as the study of analytes with very fast turn-over in brain tissue of small rodents, for example, eicosanoids, and polyphosphoinositides [33]. Acetylcholine and adenine triphosphate (ATP) are examples of small

molecules with such a fast turn-over rate that heating or freezing in situ may be needed to estimate actual levels in the living organisms [36,37]. Because of its short half-life in harvested tissue (typically much less than one minute), ATP has been used as a relative quality indicator [38]. Measurable levels of ATP indi-cate that tissue sample harvesting and stabilization has been rapid and adequate for other target analytes with half-lives in the order of minutes rather than seconds (Figure 4).

Protein denaturation & pathogen inactivationProtein denaturation is the primary mechanism behind microorganism deactivation, whether by heat or by denaturing chemicals. Viruses were shown to be inactivated by guanidine hydrochloride and gua-nidine thiocyanate [39] and by PAXgene alcohol-acid based fixation medium [40]. Bacteria rely on proteins for a host of intracellular functions necessary for their

Key term

Protein denaturation: Process by which a protein is transformed from a native, ordered, state to a less ordered state, resulting in loss of biological function, but without any change to covalent bonds.

1890 Bioanalysis (2015) 7(15)

Figure 4. Intensity images of adenine nucleotides measured by MALDI-ion mobility–MS/MS on sections (14–20 μm) of snap-frozen or heat stabilized mouse brain tissue. Sections were washed sequentially in ice-cold 70 and 95% ethanol before applying the MALDI matrix (9-aminoacridine hydrochloride, 10 mg/ml, and 0.1% TFA in 70% ethanol). Strong signals from ATP were obtained from heat-stabilized tissue, while snap-freezing resulted in virtually no signal from ATP. Reprinted with permission from [38] © Elsevier (2013).

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survival and replication. Viruses consist of RNA or DNA enveloped by proteins which are necessary for their ability to attach to and penetrate host cells. DNA and RNA are less affected by heat than is pro-tein conformation [27]. Indeed, many methods used for viral and bacterial inactivation employ heat dena-turation. The denaturation of viral proteins causes the disassembly of virus particles into noninfectious viral subunits and single proteins [41], and thermal inactivation of bacteria is associated with irrevers-ible denaturation of proteins, membranes, ribosomes and nucleic acids. Avian influenza and Newcastle dis-ease viruses have been shown to be effectively inacti-vated in homogenized whole egg and chicken meat at 60–70°C in less than 2 and 4 min, respectively [42,43]. Treatment of milk at 72°C for 20 s is a standard pro-cedure for commercial high-temperature pasteuriza-tion. Grant et al. [44] reported complete inactivation of mycobacterium paratuberculosis in milk after 25 s at 72°C. Thomas and Swayne [45] reported a pre-dicted conservative D-value (decimal reduction time) of 0.073 s at 73.9°C for the thermal inactivation of H5N1 high pathogenecity avian influenza virus in naturally infected chicken meat. Vegetative bacteria have a high water content making them sensitive to

high water activity at elevated temperature. Pathogens that withstand dry conditions require higher tempera-tures for their inactivation under dry conditions com-pared with more humid conditions [46,47], evidently because the low water content increases the stability of proteins in their native state.

Applications of heat stabilizationHeat treatment for DBS analysisFor DBS, stability of small molecules, peptides and proteins is generally improved, compared with wet blood samples, once the blood spots have been dried. However, there are sample components which will be significantly affected by enzymatic activity dur-ing the drying period. These can be stabilized by heat denaturation of proteins directly after blood spotting (Figure 5). Since such denaturation is irreversible, enzyme activity is permanent and there should be no risk that activity could be restored after rehydration of the dried sample. Only drying the blood spot will result in temporary enzyme inhibition, and there is a risk that the sample is altered by restored enzyme activity during handling of such DBS samples. Heat denaturation can be accomplished within one minute and with only minor loss of water [48]. The determina-


Figure 5. Tissue and blood sample collection with heat stabilization in a clinical or preclinical workflow prior to analysis. See [35,48] for details on heat stabilization of tissue and dried blood samples. RT: Room temperature.

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tion of oseltamivir and its active metabolite in mouse blood, spiked with oseltamivir (Figure 6), may serve as an example of DBS stabilization. Heat treatment had no apparent effect on analytical recoveries when small drug molecules were solvent extracted from DBS samples [48,49]. Very heat-sensitive compounds may not withstand the heating to about 90°C even for the short time needed for protein denaturation. Dihydroartemisinin and artemether, compounds con-taining an internal peroxide bridge and known to be chemically unstable during analysis [50], were unstable in DBS samples at 60°C and rapidly degrading at 95°C [48].

Development of quantitative assays for determina-tion of small molecules and peptides in DBS samples with good accuracy involves additional challenges in comparison to traditional plasma sample analysis. Similar to tissue sample analysis, IS cannot be added to assure a similar distribution as for the target ana-lyte in the unprocessed sample. Therefore, consistent and high recoveries in stages prior to IS addition are essential. For analytes that require sample stabilization, these requirements are emphasized. Addition of stan-dard for calibration purpose, prior to sample stabiliza-tion, will involve a risk for analyte degradation, similar but not identical to the postsampling degradation that could take place in authentic samples.

Protocols for untargeted [51–53] and targeted [54,55] proteomics have been adapted to DBS samples. Quan-titative extraction of large peptides and proteins from DBS samples prior to digestion can be a challenge, and often direct digestion can be a better alternative [55,56]. Heat denaturation of proteins prior to drying the blood spot was reported to lower extraction recovery of intact proteins [49]. But protein digestion carried out directly on the blood spot is not expected to be adversely affected, as protein denaturation (including denatur-ation by heat) is commonly used in protein digestion protocols. Digestion has been combined with immu-noaffinity enrichment for measurement of low-abun-dance protein panels using an automated SISCAPA workflow [55,57]. Results were made comparable to those from plasma sample analysis by measuring pro-teins related to erythrocytes and plasma, respectively, and using a proprietary algorithm to correct for hema-tocrit effect [55]. By including heat stabilization in the DBS sample collection procedure, the scope of

Key term

Heat stabilization: Rapid heat treatment aiming at preserving a biosample by stopping biochemical processes which would otherwise alter the sample’s composition, including primary structure and post-translational modifications of proteins.

1892 Bioanalysis (2015) 7(15)

Figure 6. Conversion of oseltamivir to oseltamivir acid in mouse blood spiked with oseltamivir. (A) Percentage of converted oseltamivir (100 × [OSC]/[OSE + OSC]) in mouse blood samples with different treatments. (B) OSE and OSC found in six replicates spotted in sequence and heat treated directly after spotting. (C) Percentage of OSE converted to OSC for six replicates plotted against storage time (time elapsed between initial spiking of blood with OSE and heat treatment of the spotted sample). OSC: Oseltamivir acid; OSE: Oseltamivir. Reprinted with permission from [48] © Denator AB (2013).

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such protocols could be expanded to phosphorylated and other short-lived proteins and peptides.

Heat stabilization for analysis of tissue samplesTissue samples can be stabilized by heat treatment either directly on the fresh sample or, in the case of a snap frozen sample, directly on the frozen sample (Figure 5), using the StabilizorTM system described in more detail elsewhere [35,34]. The tissue sample is placed in a sample container, where it is protected by a thin PTFE membrane, and placed on the tray of the Stabilizor T1 instrument. In an automated procedure, the air surrounding the sample is evacuated, the thick-ness of the sample is measured by a laser, the container with the sample is moved into the heating zone, where it contacts two heating blocks, the upper of which is lowered so that the sample is very slightly compressed for optimum heat transfer. The sample is heated for a period of time, automatically calculated from the mea-sured sample thickness, and is typically 20–50 s for a sample thickness of 2–5 mm, so that all parts of the sample reach a temperature of at least 90°C but not higher than 95°C. The stabilized sample can be stored in the evacuated sample container, where it is pro-tected from air exposure, or processed directly. Proce-

dures for heat stabilization and treatment of stabilized samples have been described in detail recently [58]. Heat stabilized tissue samples can be cryo-sectioned with the same quality as for nonstabilized tissue samples. Tissue sections are delicate, but damage dur-ing transfer to, for example, indium tin oxide (ITO) coated glass slides can be avoided by embedding the tissue in carboxymethylcellulose (CMC) prior to cryo-sectioning and using a tape transfer procedure, as was shown by Goodwin et al. (Figure 7) [59]. Initial heat sta-bilization is also compatible with subsequent chemical fixation by formalin or PAXgene reagent [Borén M, Pers.

Comm.]. For proteomic analysis, heat stabilized tissue is homogenized the same way as non-stabilized tissue. According to Kultima et al., homogenization should be slightly more rigorous than normal. Strong denatur-ing buffers, containing, for example, at least 1% SDS or 8 M urea, are needed to solubilize heat-denatured proteins [35].

Example: analysis of brain & liver homogenates – effects of heat stabilization on post-sampling release of free fatty acidsJernerén et al. [60] studied the release of free fatty acids (FFAs) as a result of exposure to room temperature


Figure 7. Photographs and MALDI–MS images of cryosections of native and heat stabilized rat brain hemispheres. (A) Photographs of rat brain hemisphere sections (20 μm) cut using a computerized cryostat microtome. Heat stabilized brain tissue was sectioned without CMC imbedding and transferred in (i) a standard way to an indium tin oxide slide or (ii) using CMC embedding and carbon tape as support. (B) MALDI–MS images of native (left) and heat-stabilized (right) rat brain sections. Intensities of ions from (i) intact PEP-19 were somewhat lower for nonstabilized tissue, while (ii & iii) intensity images of ions from two PEP-19 fragments show that these were barely detectable in heat-stabilized tissue. CMC: Carboxymethylcellulose. Reprinted with permission from [59] © Elsevier (2012).

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before organs were deep-frozen and during treatment of samples prior to instrumental analysis. Dissected organs were snap frozen, with or without previous heat stabilization, and stored at -80°C prior to analysis. Nine FFAs, ranging from myristic acid (14:0) to doco-sahexaenoic acid (22:6n-3) were measured by LC–MS after homogenization of tissue in PBS buffer and sol-vent extraction of homogenates. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs from nonstabilized liver and brain tissue compared with heat stabilized tissue. All nine FFAs were at increased levels in nonstabilized tissue.

Example: microdistribution of free sphingolipids in tissue samplesSaigusa et al. [11] developed a procedure for study of the microdistribution of sphingosine (Sph), sphinganine, sphinganinine-1-phosphate (dhS1P) and six ceramides in tissue. Such studies require lengthy sample prepa-ration including microdissection, with possible loss of sample integrity prior to downstream analysis. Ana-lyte concentrations were measured in heat-stabilized and nontreated tissue samples kept for 0–120 min prior to processing for LC–MS/MS analysis. Without heat treatment, concentrations of Sph and dhS1P were

fourfold and tenfold lower, respectively, after 15 min, while other analytes showed increasing concentrations with time, especially after 120 min after cell death had occurred. According to the authors, the signifi-cant differences in the levels of sphingolipids observed between nonstabilized and stabilized tissue indicate that the sample stabilization is important for sphingo-lipids analysis in tissue sample, and that the heat sta-bilizer (StabilizorTM T1) inactivates these enzymes but does not degrade the sphingolipids [11].

Example: identification & semiquantification of neuropeptides in mouse spinal cordBy heat stabilizing brain tissue samples, postmor-tem formation of peptides by protease activity can be avoided, which facilitates the study of native neu-ropeptides. In a recent study [10], the distribution of neuropeptides in mouse spinal cord was investigated. Dorsal and ventral halves of the cervical and lumbar enlargements were isolated under a dissecting micro-scope. Peptides were extracted and filtered through 10 kDa cut-off spin columns prior to analysis by nanoflow LC–MS/MS. In total, 43 well-characterized full-length neuropeptides and 168 previously not char-acterized peptides were identified and relatively quan-tified. The study provided evidence for involvement of



one of the peptides, [des-Ser]-cerebellin in regulation of nociceptive transmission. The peptide [des-Ser1]-cerebellin (desCER), originating from CBLN1, was identified and found to be predominantly expressed in the dorsal horn. Immunohistochemistry showed the presence of CBLN1 immunoreactivity with a punctate cytoplasmic pattern in neuronal cell bodies through-out the spinal gray matter. The signal was stronger in the dorsal compared with the ventral horn, with most CBLN1 positive cells present in outer laminae II/III, colocalizing with calbindin, a marker for excitatory interneurons. Intrathecal injection of desCER induced a dose-dependent mechanical hypersensitivity but not heat or cold hypersensitivity.

Example: detection & relative quantification of phospho-tyrosine peptides in mouse brain tissueThe effect of heat stabilization on tyrosine phosphory-lated of proteins in mouse brain tissue has been studied using antibody enrichment of phosphotyrosine peptides from digested tissue and LC–MS/MS analysis. Whole mouse brain was either heat-stabilized or snap-frozed in liquid nitrogen prior to storage at -80°C. Tissue samples were homogenized in 9 M urea, 29 mM HEPES extrac-tion buffer with protease and phosphatase inhibitors, pH 8.0. Soluble proteins were reduced, alkylated and digested with trypsin. Peptides were purified by C18 reversed phase column chromatography. Phosphotyro-sine peptides were enriched using pY1000 antibody cou-pled to protein A agarose (#9411, Cell Signaling Tech-nology, MA, USA). Released peptides were desalted and dried under vacuum prior to LC–MS/MS analysis. Measured intensities of the 1107 peptides found spanned over four orders of magnitude, with a clear trend toward higher intensities for heat-stabilized samples. For a third of the peptides, intensities were more than 50% higher, and 60 peptides were only detected in heat-stabilized samples. In contrast, only 4% of the peptides were found at more than 50% higher levels in non-stabilized samples [Cell Signaling Technologies in collaboration with

Denator AB, Unpublished Data].

Pathogen inactivationBiosampling procedures in studies involving infec-tious and pathogenic microorganisms research must be carefully considered and optimized for both exper-imental integrity and safety. Although formalin fixa-tion of such infectious tissues is often performed, the prolonged fixation protocols (14–21 days) dictated to ensure pathogen inactivation, render tissue virtu-ally unusable for proteomic analysis due to extensive protein cross-linking. The ability of heat fixation to inactivate microbial pathogens has recently be inves-

tigated [61]. Heat fixation using the Denator T1 Sta-bilizor at 95°C for 30 s was adequate for the inac-tivation of Gram negative bacteria from 3 families (Enterobacteriaceae, Moraxellaceae and Burkhold-eriaceae) in infected tissue samples. In addition to bacterial inactivation, the same heat fixation param-eters were successful in the inactivation of a +ssRNA enveloped virus (Venezuelaean Equine Encephalitis virus) in infected mouse tissue samples (brain, spleen and lung). The stabilization procedure required for inactivation did not impair tissue morphology and structure to a degree that would preclude its use for MALDI–MSI. The tissue morphology observed after heat fixation for microbial inactivation was of ade-quate integrity to obtain MALDI–MSI images and spectra of good quality, reflecting both protein and lipid molecules. This method of heat stabilization/microbial inactivation will facilitate the use of infected tissue samples for downstream proteomic, small mol-ecule drug detection and imaging MS, which in turn will advance the effort of biomarker discovery and therapeutic efficacy in infectious disease research.

Advantages & limitations of heat stabilization: comparison to other stabilization techniquesStabilization techniques not involving chemicals, such as snap-freezing and conductive heating or heat-ing by microwave irradiation have the advantage of not adding chemicals to the sample, while high con-centration buffers, detergents and organic solvents may interfere with downstream analysis. Stabilization by snap-freezing is very fast, only in situ microwave irradiation being faster, but with narrow applicabil-ity. Enzyme inhibition by freezing is temporary and exposure to enzymatic activity in the thawed sample has to be considered in the subsequent sample han-dling and analysis. The time for sample stabilization by conductive heating is in the order of 1 min, while stabilization of tissue samples by chemicals requires on one or a few hours to allow for the the chemicals to penetrate the tissue sample, unless the sample is homogenized and mixed with the stabilizing chemi-cals. Snap-freezing, stabilization by heat, and stabi-lization by chemicals thus have different applicabil-ity and should be seen as complementary rather than alternative techniques. Enzyme inhibitors also have their use, but do not provide full inhibition of tar-get enzymes and inhibit families of enzymes rather providing universal inhibition. Thus, specific inhibi-tors may be used when it is essential to keep biologi-cal activity. Table 1 summarizes characteristics of different stabilization techniques.

Degradation or alteration that takes place before the sample has been collected and heat-stabilized

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will remain a challenge. Nonenzymatic degradation will not be stopped by heat stabilization. Very heat-sensitive compounds may not withstand heating to 95°C for the short time needed for enzyme inacti-vation [48]. DNA and RNA have limited stability at high temperatures. Heat stabilization has been shown to affect RNA, measured as RNA Integrity number, while quantitative real-time PCR is still possible [34]. In some cases where proteins are denatured, the use of antibodies for enrichment of intact proteins and the application of antibody-based imaging techniques on tissue sections will be limited.

ConclusionHeat stabilization of biosamples has been used for the study of proteins and small bioactive compounds such as peptides, lipids and drug compounds. Stabi-lization can prevent degradation of unstable biomol-ecules of interest, for example, phosphorylated pro-teins, and prevent the degradation of larger molecules that could obscure the study of smaller molecules, for example, peptides and fatty acids. Heat stabilization preserves primary structure of proteins while native conformation is lost as proteins are denatured. This is adequate for molecular analysis by MS which, with a few exceptions, does not aim at studying proper-ties of proteins in their native state. The absence of additives and fixatives, which could interfere with the analysis or cause partial loss of analytes from tissue samples, makes heat stabilization compatible with mass spectrometric determination of low molecu-lar weight analytes. Inhibition of enzymatic activity should be complete and rapid and should preferably be carried out in direct conjunction with the collec-tion of samples. Heating needs to be rapid to mini-mize exposure to enzymatic activity during heating. Such heat treatment also can provide pathogen inac-tivation, heat denaturation of proteins being a com-mon mechanism for inactivation of both enzymes and microorganisms. Although rapid heat treatment has since long been used in the food industry, includ-ing high-temperature pasteurization (72°C for 15 s), its use for rapid inactivation of bacteria and viruses

in infectious biosamples is novel, and is a field for further development.

Future perspectiveClinical research for the study of molecular biomark-ers of disease requires close collaboration between experts in different fields such as pathology, molecu-lar biology and analytical chemistry. Development of instrumental techniques will continue to play a fundamental role in widening the range of biomol-ecules that can be studied and, extending the range of concentrations that can be measured toward lower concentrations, which is urgently needed considering that the majority of biomolecular species are present at extremely low concentrations. Sample stabiliza-tion techniques have a critical role in protocols for sample collection, handling and analysis designed to minimize postsampling variability. The fact that heat stabilization is an additive-free technique facili-tates its incorporation in such protocols. Today, phos-phorylated proteins are generally not considered as biomarkers due to their fast postsampling conversion rate, but this can change as improved sample collec-tion and handling protocols are implemented. For clinical diagnostics, body fluids are preferred over tis-sue samples (biopsies) whenever appropriate. Rapid additive-free stabilization of whole-blood samples in a DBS format can become an alternative to traditional plasma or serum sampling for study of the plasma proteome. The application of heat stabilization for simultaneous inactivation of pathogens is a field for further studies and development.

Financial & competing interests disclosureM Ahnoff and K Sköld are employees of Denator AB re-

search and development. K Sköld is eligible for Denator AB

stock options and has stock ownership. The authors have no

other relevant affiliations or financial involvement with any

organization or entity with a financial interest in or financial

conflict with the subject matter or materials discussed in the

manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

Executive summary

• Nonspecific protein denaturation is the common mechanism behind enzyme and pathogen inactivation by heat.

• Recently introduced pathogen inactivation by heat is very fast compared with procedures for tissue samples based on chemicals.

• Rapid heat stabilization allows for the study of sample components that would be adversely affected by enzymatic activity within the timeframe of several minutes to a few hours.

• Heat stabilization is applicable in sample collection procedures and analytical workflows for untargeted and targeted analysis of biosamples within the fields of proteins, peptides and small molecules.

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