**2. Tissue preservation**

Human tissue samples are a valuable source of information for diagnostics, therefore a lot of effort has gone into best preservation methods that minimize changes that can occur over time in storage. For example, following clinical surgery tissues need to be stored according to the protocols that minimize chemical, enzymatic, mechanical or thermal degradation and protect their molecular content. Today, tissues are most often preserved as fresh frozen (FF) or formalin-fixed paraffin-embedded (FFPE) tissues.

#### **2.1 Fresh frozen tissues**

FF tissues are obtained usually with snap freezing of tissues where the temperature achieved is below −70°C, most often in dry ice or liquid nitrogen (**Figure 1B**). To minimize variability between the samples storage and thus to minimize potential effect on molecular structure and integrity of the tissue, the European Human Frozen Tumour tissue bank (TuBa-Frost) has standardized tissue preservation by freezing in 2006 [1, 2]. An important aspect in preservation of tissues by the FF method is prevention of formation of artefacts that might result in changes to the tissue structure and morphology. For example, ice crystals that can disrupt structures within the tissue may form as a consequence of the freezing procedure due to moisture present within the tissue [3]. An alternative to snap freezing is the optimal cutting temperature (OCT) compound, which is used for tissue embedding and contains polyvinyl alcohol, polyethylene glycol (PEG) and benzalonium chloride. The OCT substance preserves tissue and enables optimal microdissection of the tissue. However, where samples will later be analyzed by mass spectrometry (MS), OCT compounds must be removed prior to analysis. This is usually achieved by washing the tissue with a special grade of alcohol or Carnoy's fluid [4] or with the use of other protocols for sample purification.

#### **2.2 Formalin-fixed paraffin-embedded tissues**

An alternative to preservation of tissue by the FF process is the use of FFPE methods (**Figure 1A**), which are routinely used by pathologists around the globe to preserve tissue by embedding in paraffin. The FFPE process preserves tissues by chemical fixation most often in 10% of formalin and is followed later by embedding in paraffin to form a tissue block for subsequent slicing. The combination of formalin fixation with paraffin embedding allows for long term storage of tissues.

**13**

**Figure 1.**

*Trends in Sample Preparation for Proteome Analysis DOI: http://dx.doi.org/10.5772/intechopen.95962*

Also, FFPE tissues are often used for histopathological studies, a routine process in examination of a patient's biopsies and clinical material [5]. Moreover, it is known that formalin leads to chemical modification of proteins in the fixed tissues causing cross-linking between proteins and modifications most often as methylation (+14 Da) as well as formation of methylene and methylol adducts to a lesser extent. As a consequence of formaldehyde induced modifications, the molecular weight or

*Overview of tissue sample processing prior to proteomic experiments. A) Tissues can be preserved and stored for long periods of time by formalin fixation and paraffin embedding which has been routinely used for decades. Proteomic analysis of FFPE tissues can be accompanied with laser capture microdissection (LCM) which helps to retrieve regions of interest on tissue sections. Further steps involve isolation of the proteins from the sections in appropriate lysis buffers and further processing for protein analysis by MS. B) Tissues can be preserved by freezing and further stored at low temperatures. Tissue should be frozen as soon as possible after retrieval and tissue can be sectioned prior to proteomic sample preparation. Tissue sections are further prepared for protein isolation, but also single cell isolation protocols could be employed to retrieve limited cell subsets prior protein extraction.*

Protein extraction and the subsequent preparation for LC–MS analysis represents

one of the key steps in proteomics (**Figure 2**). While there have been numerous protocols reported, they have mainly focused on preparation from large amounts (i.e. micrograms to milligrams) of material, which limits their utility in the study of patient clinical samples. Notably, protein extraction from FFPE preserved tissues requires removal of formaldehyde-formed cross links, which is usually carried out by heating samples in a buffered solution at an elevated temperature (95°C or 100°C). The most common buffers used for protein extraction are ammonium bicarbonate, tris(hydroxymethyl)aminomethane (Tris), and Radioimmunoprecipitation assay (RIPA) buffer. Addition of detergents to the buffer composition (e.g. sodium dodecyl sulfate (SDS), sodium dodecyl cholate (SDC), RapiGest SF surfactant™ (Waters), PPS Silent Surfactant™ (Expedeon) have been routinely used to improve protein solubilization efficiency and thus enhance protein extraction. In addition to optimization of the extraction buffers many studies also optimized other parameters like incubation time of the extraction and/or addition of various proteases to

physicochemical properties of fixed proteins can be altered.

**3. Preparation of the sample for bottom-up proteomics**

improve protein coverage during subsequent LC–MS/MS analysis.

*Trends in Sample Preparation for Proteome Analysis DOI: http://dx.doi.org/10.5772/intechopen.95962*

#### **Figure 1.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

**2. Tissue preservation**

**2.1 Fresh frozen tissues**

paraffin-embedded (FFPE) tissues.

**2.2 Formalin-fixed paraffin-embedded tissues**

challenge in their isolation and analysis. Their amphipathic nature may require use of appropriate enrichment procedures all with the goal to achieve better detection. Further, sample loss during most standard preparation procedures is inevitable, and it is even more accentuated when minute amounts of material are being processed. To minimize sample loss and thus increase sensitivity of the analysis at the MS step there have recently been several technologies developed. Specifically, improvement in technologies that allow detection of proteins down to a single cell have become available. Some of these technologies, such as nanoPOTS and micro-POTS, have already been applied to human tissues. These new possibilities to analyse small regions of tissue samples with sufficient sensitivity is opening the door to many applications such as profiling of selected regions of a tumorous zone or detection of proteins from subcellular populations. These new applications aimed at working with 1 to 100s or 1000s of cells will likely have increasing importance in

clinics, but only if they can be developed into routine and robust methods.

Human tissue samples are a valuable source of information for diagnostics, therefore a lot of effort has gone into best preservation methods that minimize changes that can occur over time in storage. For example, following clinical surgery tissues need to be stored according to the protocols that minimize chemical, enzymatic, mechanical or thermal degradation and protect their molecular content. Today, tissues are most often preserved as fresh frozen (FF) or formalin-fixed

FF tissues are obtained usually with snap freezing of tissues where the temperature

achieved is below −70°C, most often in dry ice or liquid nitrogen (**Figure 1B**). To minimize variability between the samples storage and thus to minimize potential effect on molecular structure and integrity of the tissue, the European Human Frozen Tumour tissue bank (TuBa-Frost) has standardized tissue preservation by freezing in 2006 [1, 2]. An important aspect in preservation of tissues by the FF method is prevention of formation of artefacts that might result in changes to the tissue structure and morphology. For example, ice crystals that can disrupt structures within the tissue may form as a consequence of the freezing procedure due to moisture present within the tissue [3]. An alternative to snap freezing is the optimal cutting temperature (OCT) compound, which is used for tissue embedding and contains polyvinyl alcohol, polyethylene glycol (PEG) and benzalonium chloride. The OCT substance preserves tissue and enables optimal microdissection of the tissue. However, where samples will later be analyzed by mass spectrometry (MS), OCT compounds must be removed prior to analysis. This is usually achieved by washing the tissue with a special grade of alcohol or Carnoy's fluid [4] or with the use of other protocols for sample purification.

An alternative to preservation of tissue by the FF process is the use of FFPE methods (**Figure 1A**), which are routinely used by pathologists around the globe to preserve tissue by embedding in paraffin. The FFPE process preserves tissues by chemical fixation most often in 10% of formalin and is followed later by embedding in paraffin to form a tissue block for subsequent slicing. The combination of formalin fixation with paraffin embedding allows for long term storage of tissues.

**12**

*Overview of tissue sample processing prior to proteomic experiments. A) Tissues can be preserved and stored for long periods of time by formalin fixation and paraffin embedding which has been routinely used for decades. Proteomic analysis of FFPE tissues can be accompanied with laser capture microdissection (LCM) which helps to retrieve regions of interest on tissue sections. Further steps involve isolation of the proteins from the sections in appropriate lysis buffers and further processing for protein analysis by MS. B) Tissues can be preserved by freezing and further stored at low temperatures. Tissue should be frozen as soon as possible after retrieval and tissue can be sectioned prior to proteomic sample preparation. Tissue sections are further prepared for protein isolation, but also single cell isolation protocols could be employed to retrieve limited cell subsets prior protein extraction.*

Also, FFPE tissues are often used for histopathological studies, a routine process in examination of a patient's biopsies and clinical material [5]. Moreover, it is known that formalin leads to chemical modification of proteins in the fixed tissues causing cross-linking between proteins and modifications most often as methylation (+14 Da) as well as formation of methylene and methylol adducts to a lesser extent. As a consequence of formaldehyde induced modifications, the molecular weight or physicochemical properties of fixed proteins can be altered.
