2 - Sample preparation.docx

# Sample preparation for proteomics Sample preparation for proteomics is a critical multi-step process that aims to extract, solubilize, and purify proteins from biological samples in a manner suitable for downstream proteomic analysis. The ultimate goal is to maximize the recovery of proteins while minimizing degradation and the introduction of artifacts, thereby ensuring accurate and comprehensive proteomic profiling. ## 1. Sample preparation for proteomics Scientific questions and experimental design form the foundation of any proteomic study, guiding the subsequent choices in sample collection and preparation. Key considerations include the specific research question (e.g., investigating a single protein, proteoforms, or the entire proteome), the availability of suitable techniques, and whether tertiary structures or protein complexes need to be preserved. ### 1.1 Sample collection The diversity of biological samples necessitates careful consideration during collection to ensure representativeness and minimize degradation. #### 1.1.1 Sample types * **Cell lines:** Offer controlled laboratory settings but may not always reflect the complexity of real tissues or cells. * **Patient samples:** Exhibit higher variability due to differences in handling and patient factors. The timing of sample stabilization (e.g., freezing) is a crucial variable. Analyzing large sample numbers can enhance statistical significance. * **Serum and plasma:** Can be highly variable and challenging to analyze due to significant differences in protein concentrations. * **Biobanked samples:** Can show substantial variability, particularly formalin-fixed paraffin-embedded (FFPE) tissues. #### 1.1.2 Sample degradation Minimizing sample degradation is paramount, especially for cells, tissues, and organs. Proteins are less stable than DNA. Stress can induce proteolytic and phosphatase activity, leading to the rapid disappearance of phosphorylated peptides. Degradation can be inhibited by: * **Inhibitors:** Adding protease, phosphatase inhibitors, etc. * **Denaturation:** Employing chaotropic agents or detergents. * **Temperature:** Cold temperatures can inhibit activity reversibly, while heating usually causes irreversible denaturation. * **pH adjustment:** Modifying the pH can also inhibit enzymatic activity. ### 1.2 Protein extraction and solubilization The primary aim of protein extraction is to disrupt cellular structures (membranes, cell walls) and release proteins into a soluble solution. Completeness of extraction is the ideal but often unachievable goal. #### 1.2.1 Extraction methods Methods vary significantly based on the sample type and the desired outcome, with a focus on preventing protein degradation during lysis. ##### 1.2.1.1 Soft extraction methods These methods aim to lyse cells with minimal damage to proteins. * **Osmotic shock:** * **Mechanism:** Cells are placed in a solution with a significantly different osmotic pressure, causing water to rapidly enter or exit the cell, leading to rupture. This is achieved by alternating between hypertonic and hypotonic environments or by cycles of freezing and thawing, which alter the solute-to-water ratio. * **Application:** Primarily used for cells with less robust walls, such as animal or certain algal cells. * **Detergents:** * **Mechanism:** Amphipathic molecules that disrupt cell membranes by forming micelles with membrane lipids. * **Types:** * **Anionic detergents (e.g., SDS):** Disrupt membranes, protein-protein interactions, and protein activity. SDS can denature proteins into rigid rods proportional to their molecular weight. Binding is cooperative. * **Non-ionic detergents:** Considered non-denaturing, breaking lipid-lipid and lipid-protein interactions. Widely used for isolating membrane proteins in their active form. * **Zwitterionic detergents:** Possess a net zero charge, disrupting protein-protein interactions. Used in isoelectric focusing and ion exchange chromatography. * **Consideration:** Detergents often need to be removed prior to digestion or LC-MS analysis via dialysis, gel filtration, mass-cut-off filters, or protein precipitation. * **Enzymatic digestion:** * **Mechanism:** Enzymes like lysozyme break down specific components of cell walls, such as the β-1,4-linkage in peptidoglycans of bacteria. * **Application:** Primarily used for bacteria. * **Dounce homogenizer:** * **Mechanism:** A glass pestle moves within a glass tube, creating shear stress that breaks cells with minimal heating. This method can keep organelles intact. * **Advantage:** Minimal heating, preservation of organelles. ##### 1.2.1.2 Harsh extraction methods These methods employ more aggressive physical forces to break open tougher cell structures. * **Blender, tissue chopper, and cryo-grinding:** * **Mechanism:** Samples are frozen (often in liquid nitrogen) and then mechanically disrupted using a pestle and mortar (cryo-grinding) or by blending/chopping. * **Application:** Commonly used for lysing organs. * **Bead beater:** * **Mechanism:** Samples are agitated at high speed with small beads (glass, ceramic, or steel), causing physical disruption of cell walls and membranes through collisions. * **Application:** Effective for breaking open cells like bacteria, yeast, fungi, and plant cells. * **Sonication:** * **Mechanism:** High-frequency vibrations create microscopic vapor bubbles in the suspension. The rapid formation and implosion of these bubbles generate intense local shock waves, whose shear forces rupture cell membranes and walls. * **Consideration:** Samples should be kept on ice between pulses to prevent overheating. #### 1.2.2 Protein solubilization The goal of solubilization is to achieve maximum solubility for the widest range of proteins. * **Chaotropic agents:** * **Mechanism:** These agents (e.g., urea, thiourea, guanidinium chloride) disrupt the water network by forming stronger interactions with water molecules. This reduces the energetic penalty of exposing hydrophobic regions of proteins to the aqueous environment, thereby weakening the driving force for protein aggregation and causing insoluble protein aggregates to dissociate. * **Types and applications:** * **Urea (7-8 M):** Efficient hydrogen bond disruption, suitable for 2D-PAGE, but less effective for hydrophobic disruption. * **Thiourea (2 M):** Good for hydrophobic disruption, beneficial for membrane proteins. * **Guanidinium chloride (6 M):** Effective for both hydrogen bond and hydrophobic region disruption. ### 1.3 Reduction and alkylation Proteins can be stabilized by covalent disulfide bonds, which must be broken before digestion for LC-MS analysis. * **Reducing agents:** β-mercaptoethanol or dithiothreitol (DTT) are commonly used to break disulfide bridges. * **Alkylation:** Following reduction, the free thiols are typically alkylated to prevent disulfide bond reformation. Alkylating agents like iodoacetamide are often used. ### 1.4 Protein digestion Proteins are typically digested into peptides using specific proteases. * **Trypsin:** The most common protease, cleaving proteins into peptides of approximately 10-20 amino acids in length, which are ideal for LC-MS. Trypsin cleaves C-terminal to lysine (Lys) or arginine (Arg) residues, which are favorable for ionization. Digestion usually occurs overnight at 37°C. * **Lysyl endopeptidase C (LysC):** Can be combined with trypsin for more complete digestion. ### 1.5 Peptide purification or selection Various compounds introduced during sample preparation (detergents, salts, protease inhibitors) need to be removed before LC-MS analysis. #### 1.5.1 On protein level * **Precipitation (e.g., acetone precipitation):** * **Mechanism:** Proteins are precipitated by adding cold acetone to the sample, followed by incubation and centrifugation. The supernatant is discarded, and the protein pellet is dried and resolubilized. * **Consideration:** Avoid over-drying the pellet, as it may become difficult to dissolve. * **Gel filtration:** * **Mechanism:** Proteins are passed through a column packed with porous beads. Larger proteins (proteins) elute faster than smaller buffer contaminants as they are excluded from the pores. * **Format:** Available in column or spin tube formats. * **Filtration (Molecular weight cut-off filters):** * **Mechanism:** Filters with specific molecular weight cut-offs (e.g., 3, 5, 10, 30 kDa) are used to separate proteins from smaller molecules through centrifugation. * **Advantage:** Easy and quick. * **SDS-PAGE:** * **Mechanism:** Separates proteins based on molecular weight and can effectively remove detergents, salts, and protease inhibitors. #### 1.5.2 On peptide level * **C18 solid phase extraction (SPE):** * **Mechanism:** A chromatographic method that separates peptides based on hydrophobicity. The stationary phase is typically silica beads with covalently attached long alkyl chains (C18). Hydrophobic peptides bind to the stationary phase, while hydrophilic molecules pass through. Elution is achieved with organic solvents. * **Steps:** Column flushing with organic solvent, followed by aqueous buffer, sample loading, washing with aqueous buffer to remove impurities, and finally elution with a strong organic solvent. * **Ion exchange chromatography:** * **Mechanism:** Separates molecules based on their net surface charge. ### 1.6 Prefractionation For highly complex peptide samples where standard RP-HPLC-MS may not provide sufficient depth, prefractionation adds an additional separation step orthogonal to the final LC-MS. * **High pH RP-HPLC:** * **Mechanism:** This method uses reverse-phase chromatography at a high pH (whereas standard LC-MS typically uses low pH). This orthogonal separation strategy can significantly simplify the peptide mixture before the final analysis. * **Strong cation exchange (SCX) chromatography:** * **Mechanism:** Separates peptides based on their charge. This is often used as an orthogonal dimension to RP-HPLC. --- # Protein extraction methods Protein extraction is a critical initial step in proteomics that aims to release proteins from their cellular or tissue matrix into a soluble form suitable for downstream analysis. The choice of method depends heavily on the sample type, the specific proteins of interest, and the desired preservation of protein structure and interactions. ### 2.1 Principles of protein extraction The overarching goal of protein extraction is to achieve the maximum possible release and solubilization of proteins while minimizing degradation and denaturation. This process involves disrupting cellular structures (cell membranes and, if present, cell walls) to bring proteins into solution. #### 2.1.1 Preventing protein degradation Proteins are susceptible to degradation by endogenous enzymes. To prevent this, especially during cell lysis, it is crucial to: * Inhibit proteolytic and phosphatase activity using **protease and phosphatase inhibitor cocktails**. * Denature proteins using **chaotropic agents or detergents**. * Reduce enzymatic activity through **temperature control** (cold can be reversible, while heating is usually irreversible). * Adjust the **pH** of the solution. #### 2.1.2 Disruption methods Methods for disrupting cells can be broadly categorized as mechanical and non-mechanical, with varying degrees of "softness" or "harshness" which can influence protein denaturation. ##### 2.1.2.1 Mechanical methods These methods rely on physical forces to break cells. * **Dounce homogenizer:** * **Mechanism:** A pestle is moved up and down within a glass tube, creating shear stress in the narrow space between the pestle and tube wall, which lyses cells. * **Advantages:** Minimal heating and can preserve organelles like the nucleus. * **Blender, tissue chopper, and cryo-grinding:** * **Mechanism (Cryo-grinding):** Samples are flash-frozen in liquid nitrogen and then disrupted in a solid state using a pestle and mortar. * **Application:** Primarily used for lysing solid tissues and organs. * **Bead beater:** * **Mechanism:** Cells are mixed with small beads (glass, ceramic, or steel) in a vial. The vial is then agitated at high speed, causing the beads to collide with each other and the cells, leading to cell lysis through mechanical stress. * **Application:** Effective for robust cells like bacteria, yeast, fungi, and plant cells. * **Sonication:** * **Mechanism:** A sonicator probe vibrates at high frequencies in a cell suspension, creating microscopic vapor bubbles. The rapid formation and implosion of these bubbles generate intense local shock waves and shear forces that rupture cell membranes and walls. * **Note:** It is important to keep the sample on ice between sonication pulses to prevent overheating. * **Freeze-thawing:** * **Mechanism:** Freezing reduces the amount of liquid water, creating a hyperosmotic environment. Upon thawing, the liquid water increases, making the environment hypoosmotic. Water rushes into the cells, causing them to burst. ##### 2.1.2.2 Non-mechanical methods These methods use chemical or enzymatic agents to disrupt cells. * **Osmotic shock (soft method):** * **Mechanism:** Cells are placed in a solution with significantly different osmotic pressure. Rapid transfer between solutions of opposite tonicity (hypertonic to hypotonic or vice versa) causes a sudden influx or efflux of water, creating pressure that ruptures the cell membrane. * **Application:** Best suited for cells with less robust cell walls, such as animal cells. * **Detergents (soft method):** * **Mechanism:** Detergents are amphipathic molecules that disrupt the lipid bilayer of cell membranes by forming micelles. Their hydrophobic tails insert into the lipid core, while their hydrophilic heads interact with water, leading to membrane breakdown and solubilization of membrane proteins. * **Types of detergents:** * **Anionic detergents (e.g., SDS):** Disrupt membranes, protein-protein interactions, and protein activity. They tend to alter proteins into rigid rods proportional to their molecular weight, which is useful for SDS-PAGE. * **Non-ionic detergents:** Considered non-denaturing as they primarily disrupt lipid-lipid and lipid-protein interactions. Widely used for isolating membrane proteins in their active form. * **Zwitterionic detergents:** Possess both positive and negative charges, resulting in a net zero charge. They disrupt protein-protein interactions and are useful in isoelectric focusing and ion exchange chromatography. * **Note:** Detergents often need to be removed prior to downstream analyses (e.g., digestion, LC-MS) via dialysis, gel filtration, or mass cut-off filters. * **Enzymatic digestion (soft method):** * **Mechanism:** Enzymes like lysozyme are used to degrade specific components of cell walls, such as the $\beta$-1,4 linkages between NAM and NAG in bacterial peptidoglycan. * **Application:** Primarily used for bacteria. ### 2.2 Protein solubilization The aim of solubilization is to bring as many proteins as possible into solution for analysis. #### 2.2.1 Chaotropic agents Chaotropic agents disrupt the hydrogen bond network of water, reducing the hydrophobic effect that drives protein aggregation. By making it easier for water to interact with hydrophobic surfaces, they weaken the forces that cause denatured proteins to clump together. * **Urea (7-8 M):** Efficient at disrupting hydrogen bonds, but less effective for hydrophobic interactions. Useful for 2D-PAGE. * **Thiourea (2 M):** Good for hydrophobic disruption, particularly beneficial for membrane proteins. * **Guanidinium chloride (6 M):** Effective for both hydrogen bond and hydrophobic region disruption. ### 2.3 Reduction and alkylation Disulfide bridges ($S-S$) covalently stabilize protein structures. These bonds must be broken before protein digestion and LC-MS analysis. * **Reducing agents:** $\beta$-mercaptoethanol or dithiothreitol (DTT) are commonly used to break disulfide bonds. * **Alkylation:** Following reduction, alkylation can be performed to prevent the reformation of disulfide bonds. ### 2.4 Protein digestion Protein digestion, typically using **trypsin**, breaks down proteins into smaller peptides (10-20 amino acids in length), which are ideal for LC-MS analysis due to their favorable ionization properties. Trypsin cleaves specifically after lysine (Lys) or arginine (Arg) residues. Digestion is usually performed overnight at $37^\circ\text{C}$. For more complete digestion, proteases like LysC can be used in combination with trypsin. ### 2.5 Peptide purification or selection Contaminants such as detergents, salts, and protease inhibitors must be removed from protein or peptide samples before digestion or LC-MS. #### 2.5.1 Protein-level purification * **Acetone precipitation:** Proteins are precipitated by adding cold acetone to the sample, followed by centrifugation. The supernatant is discarded, and the protein pellet is dried and resolubilized. * **Gel filtration:** Proteins are passed through a column packed with porous beads. Larger proteins elute faster as they cannot enter the pores, while smaller molecules are retained longer. This method separates proteins from buffer contaminants. * **Filtration (Molecular weight cut-off filters):** Various filters with specific molecular weight cut-offs (e.g., 3, 5, 10, 30 kDa) can be used to separate proteins from smaller molecules in a single centrifugation step. * **SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis):** Effectively removes detergents, salts, and protease inhibitors while also separating proteins based on their molecular weight. #### 2.5.2 Peptide-level purification * **C18 solid-phase extraction (SPE):** * **Mechanism:** This is a chromatographic technique that separates peptides based on hydrophobicity. The stationary phase consists of silica beads with long C18 hydrocarbon chains, making it highly hydrophobic. Hydrophobic peptides bind to the stationary phase, while hydrophilic molecules pass through. Elution is achieved using increasing concentrations of organic solvent. * **Application:** Used to purify and concentrate peptides. #### 2.5.3 Prefractionation For highly complex peptide samples, prefractionation steps can be employed before LC-MS to increase analytical depth. * **High pH reversed-phase HPLC (RP-HPLC):** This technique is orthogonal to the low pH RP-HPLC typically used in LC-MS. It separates peptides based on hydrophobicity at a high pH. * **Strong cation exchange (SCX) chromatography:** Separates peptides based on their charge. --- # Protein solubilization and degradation prevention This section focuses on essential techniques for maximizing protein solubility after extraction and preventing sample degradation during processing. ### 3.1 Preventing protein degradation Protein degradation must be minimized, particularly in complex biological samples like cells and tissues. This degradation can be initiated by various stresses, which can activate endogenous enzymes like proteases and phosphatases. Phosphorylated peptides are especially susceptible to rapid degradation. #### 3.1.1 Strategies for inhibition of degradation Enzymatic degradation can be prevented through several methods: * **Inhibitors:** The addition of specific inhibitors, such as protease and phosphatase inhibitors, can effectively block enzyme activity. * **Denaturation:** Employing agents that denature proteins, like chaotropic agents or detergents, can also inhibit enzymatic activity by altering enzyme structure. * **Temperature:** Lowering the temperature (cold) can reversibly slow down enzymatic reactions. Heating generally leads to irreversible denaturation and inactivation of enzymes. * **pH adjustment:** Modifying the pH of the sample can also inactivate enzymes by altering their optimal functional conditions. ### 3.2 Protein extraction and solubilization The primary goal of protein extraction is to release as many proteins as possible into solution, aiming for completeness, although absolute completeness is rarely achieved. Different methods are employed based on the sample type and research objective, ranging from soft to harsh techniques. #### 3.2.1 Soft extraction methods Soft methods aim to disrupt cells with minimal denaturation or damage to protein structures. * **Osmotic shock:** This method is suitable for cells with less robust walls, such as animal cells. It involves rapidly changing the surrounding salt concentration to induce water movement into or out of the cell, ultimately causing the cell membrane to rupture. This can also be achieved through freeze-thaw cycles, where freezing creates a hyperosmotic environment within the remaining liquid water, and thawing leads to a hypotonic environment that causes cell lysis. * **Detergents:** Detergents are amphipathic molecules that disrupt lipid-lipid and lipid-protein interactions within cell membranes, leading to membrane solubilization. * **Anionic detergents:** These, like SDS (sodium dodecyl sulfate), have anionic head groups and are effective at disrupting membranes and protein-protein interactions. SDS can alter proteins into rigid rods proportional to their molecular weight through cooperative binding. * **Non-ionic detergents:** These are generally considered non-denaturing as they primarily disrupt lipid interactions without significantly affecting protein structure. They are useful for isolating membrane proteins while maintaining their activity. * **Zwitterionic detergents:** These possess a net zero charge and can disrupt protein-protein interactions. They are used in techniques like isoelectric focusing and ion exchange chromatography. Detergents often need to be removed before downstream analyses like digestion or LC-MS, which can be achieved through dialysis, gel filtration, mass cut-off filters, or protein precipitation. * **Enzymatic digestion:** This method is primarily used for bacterial lysis. Enzymes like lysozyme can degrade the peptidoglycan layer of bacterial cell walls, leading to cell rupture. * **Dounce homogenizer:** This mechanical method uses a glass pestle within a glass tube to shear cells. It offers minimal heating and can preserve organelles, making it suitable for samples where structural integrity is important. #### 3.2.2 Harsh extraction methods Harsh methods employ more forceful disruption techniques, often used for tougher samples like organs. * **Blender, tissue chopper, and cryo-grinding:** These methods are typically applied to organs. Cryo-grinding involves flash-freezing the sample in liquid nitrogen and then disrupting its solid state using a mortar and pestle. * **Bead beater:** This technique involves mixing the sample with small beads (glass, ceramic, or steel) within a vial. The vial is then subjected to high-speed shaking, causing the beads to collide and lyse cells through mechanical stress. Care must be taken as some proteins can bind to metal beads. * **Sonication:** High-frequency vibrations are used to create microscopic vapor bubbles in a cell suspension. The rapid formation and implosion of these bubbles generate shock waves that rupture cell membranes and walls. It is important to keep the sample on ice between sonication pulses to prevent overheating. #### 3.2.3 Protein solubilization goals and strategies The objective of protein solubilization is to achieve maximum solubility for the widest possible range of proteins. * **Chaotropic agents:** These agents, such as urea (7–8 M), thio-urea (2 M), and guanidinium chloride (6 M), are crucial for counteracting the hydrophobic effect, a major driver of protein aggregation. Proteins tend to fold with hydrophobic regions buried internally. When proteins denature, these hydrophobic regions become exposed to the aqueous environment, leading to aggregation to minimize unfavorable interactions with water. Chaotropic agents disrupt the hydrogen-bonding network of water, making it more favorable for water to interact with non-polar surfaces. This reduces the driving force for aggregation, causing aggregated proteins to dissociate. * **Urea:** Effective at disrupting hydrogen bonds but less so for hydrophobic interactions. * **Thio-urea:** Good for hydrophobic disruption, particularly useful for membrane proteins. * **Guanidinium chloride:** Effective for both hydrogen bond and hydrophobic region disruption. ### 3.3 Protein degradation prevention during processing Beyond immediate lysis, maintaining protein integrity throughout the workflow is critical. #### 3.3.1 Reduction and alkylation Disulfide bridges are covalent bonds that stabilize protein tertiary structure. These bonds must be broken prior to digestion and LC-MS analysis. This is commonly achieved using reducing agents such as $\beta$-mercaptoethanol or dithiothreitol (DTT). Following reduction, alkylation is performed to prevent the re-formation of disulfide bonds. #### 3.3.2 Protein digestion Protein digestion is typically performed using the enzyme trypsin, which cleaves proteins into peptides of approximately 10–20 amino acids long. These peptide lengths are ideal for LC-MS analysis as they are easily ionized. Trypsin cleaves after lysine (Lys) and arginine (Arg) residues, which are favorable for ionization. Digestion is usually carried out overnight at 37°C. For more comprehensive digestion, it can be combined with other proteases like LysC. Digestion can often proceed in the presence of up to 2 M urea. #### 3.3.3 Peptide purification or selection Various compounds, including detergents, salts, and protease inhibitors, must be removed before digestion or LC-MS analysis to avoid interference. * **On the protein level:** Methods include precipitation (e.g., acetone precipitation), gel filtration, filtration using molecular weight cut-off membranes, SDS-PAGE, and dialysis. * **Acetone precipitation:** Involves precipitating proteins by adding cold acetone, followed by centrifugation, removal of the supernatant, and evaporation of residual acetone. It is crucial not to over-dry the pellet to ensure proper resolubilization. * **Gel filtration:** Separates proteins from buffer contaminants based on size using porous beads. Larger proteins pass through the column faster than smaller molecules. This can also be performed in a spin-tube format. * **Filtration:** Molecular weight cut-off filters (e.g., 3, 5, 10, 30 kDa) offer a quick method for size-based separation through centrifugation. * **On the peptide level:** Techniques like C18 solid-phase extraction (SPE) and ion exchange chromatography are employed. * **C18 solid-phase extraction:** This is a chromatographic method that separates peptides based on hydrophobicity. The stationary phase, typically silica beads with covalently attached C18 chains, is highly hydrophobic. Hydrophobic molecules bind to the stationary phase, while hydrophilic molecules pass through. Elution is achieved using a strong organic solvent. This method also aids in concentrating the peptide sample. * **High pH RP-HPLC:** When peptide samples are extremely complex, and standard RP-HPLC coupled with MS is insufficient to achieve the desired depth of proteomic analysis, prefractionation using an additional orthogonal LC step is beneficial. High pH reversed-phase HPLC can be used before the standard low pH RP-HPLC typically employed for LC-MS. --- # Peptide purification and prefractionation Peptide purification and prefractionation are critical steps in proteomics workflows designed to simplify complex peptide mixtures and remove interfering substances before LC-MS analysis, thereby enhancing analytical depth and accuracy. ### 4.1 Peptide purification Peptide purification aims to remove contaminants such as detergents, salts, protease inhibitors, and buffer components that can interfere with downstream LC-MS analysis. These purification methods can be applied at either the protein or peptide level. #### 4.1.1 Protein-level purification Purification at the protein level can be achieved through several methods: * **Acetone precipitation:** This is a common method where proteins are precipitated by adding cold acetone to the sample. The principle relies on the reduced solubility of proteins in organic solvents like acetone, causing them to aggregate and pellet. > **Tip:** Ensure the acetone is sufficiently cold (e.g., -20°C) for optimal precipitation. Do not over-dry the protein pellet after acetone evaporation, as it may become difficult to re-dissolve. * **Gel filtration chromatography:** This technique separates molecules based on their size. Proteins, being larger than most buffer contaminants, pass through a column packed with porous beads at different rates, allowing for separation. Spin column formats are also available for easier use. * **Filtration using molecular weight cutoff (MWCO) filters:** These filters allow for quick separation of molecules based on their size. By selecting a filter with an appropriate MWCO, larger molecules like proteins can be retained while smaller contaminants pass through, or vice-versa, typically in a single centrifugation step. * **SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis):** While primarily a separation technique, SDS-PAGE can effectively remove detergents, salts, and protease inhibitors. It also separates proteins based on their molecular weight. #### 4.1.2 Peptide-level purification Purification methods applied after protein digestion yield purified peptides: * **C18 solid-phase extraction (SPE):** This is a highly effective chromatographic method for separating peptides based on their hydrophobicity. The stationary phase consists of silica beads covalently bonded with long C18 (octadecyl) chains, creating a hydrophobic surface. * **Mechanism:** The C18 column is first equilibrated with a polar solvent (e.g., water-based buffer with a small amount of organic solvent). Hydrophobic peptides in the sample will bind to the hydrophobic stationary phase. Less hydrophobic molecules and contaminants will pass through. The column is then washed with a more aqueous buffer to remove weakly bound impurities. Finally, bound peptides are eluted using a strong organic solvent (e.g., methanol or acetonitrile) that disrupts the hydrophobic interactions. The collected liquid is the eluate containing purified and concentrated peptides. > **Tip:** C18 SPE is crucial for removing salts and other polar contaminants that can suppress ionization during MS analysis. * **Ion exchange chromatography:** This method separates molecules based on their charge. Peptides can be bound to a stationary phase with opposite charge and then eluted by changing the pH or ionic strength of the mobile phase. This technique is often used as an orthogonal separation to RP-HPLC. ### 4.2 Prefractionation Prefractionation strategies are employed to simplify highly complex peptide samples that would otherwise be inadequately resolved by a single LC-MS run, thus increasing the depth of proteome coverage. These methods introduce an additional separation step that is orthogonal to the final LC-MS step. #### 4.2.1 High pH reversed-phase HPLC (RP-HPLC) High pH RP-HPLC is a common prefractionation technique. The primary LC-MS analysis typically uses low pH mobile phases. By performing an initial separation at high pH, an orthogonal separation mechanism is achieved. This method separates peptides based on their hydrophobicity, similar to standard RP-HPLC, but under different pH conditions, leading to a different separation selectivity. #### 4.2.2 Strong cation exchange (SCX) chromatography SCX chromatography is another widely used prefractionation technique. It separates peptides based on their charge. Peptides are bound to a negatively charged stationary phase and then eluted by increasing the salt concentration of the mobile phase. SCX provides a separation mechanism that is orthogonal to RP-HPLC, which separates based on hydrophobicity. > **Example:** A typical prefractionation workflow might involve first separating peptides using SCX chromatography into several fractions. Each of these SCX fractions is then individually subjected to a second dimension of separation using high pH RP-HPLC, further reducing complexity. Finally, each of these more simplified fractions is analyzed by standard low pH RP-LC-MS/MS. --- ## Common mistakes to avoid - Review all topics thoroughly before exams - Pay attention to formulas and key definitions - Practice with examples provided in each section - Don't memorize without understanding the underlying concepts

| Term | Definition | |------|------------| | Proteomics | The large-scale study of proteins, particularly their structures and functions. It aims to systematically study the entire set of proteins produced or modified by an organism or system under specific conditions. | | Proteoforms | Variations of protein sequences arising from genetic mutations, alternative splicing, or post-translational modifications. Understanding proteoforms is crucial for a complete picture of the proteome. | | Proteome | The entire complement of proteins, including their isoforms and post-translational modifications, that is produced by an organism, cell, or biological system at a given time and under specific conditions. | | Sample collection | The process of gathering biological material for analysis, which requires careful handling to minimize degradation and maintain the integrity of proteins and other biomolecules. | | Protein extraction | The process of releasing proteins from their cellular or tissue environment into a soluble form that can be further analyzed. This typically involves disrupting cell membranes and walls. | | Solubilization | The process of dissolving a substance, in this context, proteins, into a liquid medium. Effective solubilization is essential for downstream analyses. | | Protein separation or purification | Techniques used to isolate specific proteins or classes of proteins from complex mixtures, often based on their physical or chemical properties. | | Reduction and alkylation | Chemical treatments applied to proteins to break disulfide bonds (reduction) and prevent their re-formation (alkylation), which is necessary for subsequent digestion and analysis. | | Digestion | The enzymatic breakdown of proteins into smaller peptide fragments. This is typically done using proteases like trypsin to prepare samples for mass spectrometry. | | Peptide purification or selection | Methods used to clean up and concentrate peptide mixtures after digestion, removing unwanted contaminants that can interfere with analytical techniques. | | Prefractionation | A strategy employed to reduce the complexity of a sample before analysis, often involving multiple chromatographic steps, to increase the depth of proteomic coverage. | | Research question | The central inquiry that drives a scientific investigation, guiding experimental design and the selection of appropriate methodologies. | | Tertiary structure | The three-dimensional folding of a single polypeptide chain, including alpha-helices, beta-sheets, and loops, stabilized by various interactions like hydrogen bonds and hydrophobic interactions. | | Protein complexes/interactions | Assemblies of two or more proteins that function together, or the transient associations between proteins, which are vital for cellular processes. | | Proteomics sample preparation protocols | A series of steps designed to prepare biological samples for proteomic analysis, often tailored to specific research questions and sample types. | | Cell lines | Cultures of cells derived from multicellular organisms that can be maintained and grown in vitro under controlled laboratory conditions. | | Patient samples | Biological specimens obtained directly from individuals, which can be more variable due to physiological differences and handling conditions. | | Serum and plasma | Blood components used for analysis; serum is plasma without clotting factors, while plasma contains clotting factors. Both are rich sources of proteins but can be highly variable. | | Biobanked samples | Biological samples that have been collected, processed, and stored for future research purposes, potentially over long periods. | | FFPE tissues (formalin-fixed paraffin embedded) | Tissues that have been preserved by fixation with formalin and embedded in paraffin wax for long-term storage and histological examination. This can impact protein integrity. | | Sample degradation | The breakdown of biomolecules, such as proteins, due to enzymatic activity, environmental factors, or improper storage, leading to loss of sample quality. | | Proteolytic activity | The enzymatic breakdown of proteins by proteases. This must be inhibited during sample preparation to prevent sample degradation. | | Phosphatase activity | The enzymatic removal of phosphate groups from proteins. This activity needs to be inhibited to preserve phosphorylated peptides. | | Phosphorylated peptides | Peptide fragments that contain one or more phosphate groups attached to amino acid residues. These are important indicators of cellular signaling. | | Protease inhibitor cocktail | A mixture of chemical compounds designed to inhibit the activity of various proteases, thus preventing protein degradation. | | Denaturation | The process by which a protein loses its native three-dimensional structure, often due to exposure to heat, extreme pH, or chemical agents, which can lead to loss of function. | | Chaotropic agents | Chemical compounds that disrupt the structure of water and interfere with hydrogen bonding, thereby reducing the hydrophobic effect and promoting protein unfolding or solubilization. Examples include urea and guanidinium chloride. | | Detergents | Amphipathic molecules that can solubilize lipids and hydrophobic proteins by forming micelles. They are classified as anionic, non-ionic, or zwitterionic. | | pH | A measure of the acidity or alkalinity of a solution. Adjusting pH can be used to control enzymatic activity or protein solubility. | | Cell membrane | The selectively permeable outer boundary of animal cells and the inner boundary of plant and bacterial cells, composed of a lipid bilayer. | | Cell wall | A rigid outer layer surrounding the plasma membrane of plant cells, fungi, and bacteria, providing structural support and protection. | | Solubilize | To cause a solute to dissolve in a solvent, making it form a solution. | | Completeness | In protein extraction, the ideal goal of releasing all target proteins from the sample, though perfect completeness is often unattainable. | | Mechanical lysis | A method of breaking open cells using physical force, such as sonication, bead beating, or homogenization. | | Non-mechanical lysis | Methods of breaking open cells that do not rely on physical force, such as using detergents or enzymatic digestion. | | Artefacts | Unintended structures or changes introduced into a sample during preparation or analysis that can lead to misinterpretation of results. | | Sonication | A technique that uses high-frequency sound waves to disrupt cells and tissues, creating shock waves that rupture cell membranes. | | Bead beating | A method of cell lysis where beads are agitated at high speed within a sample, causing mechanical stress that breaks open cells. | | Mixing | Simple agitation used in some lysis protocols, often in conjunction with other methods. | | Freeze thawing | A process of repeatedly freezing and thawing a sample to induce cell lysis due to the formation and melting of ice crystals. | | Detergents | Amphipathic molecules with a hydrophilic head and a hydrophobic tail, used to disrupt cell membranes and solubilize proteins. | | Amphipathic molecules | Molecules that contain both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions, allowing them to interact with both water and lipids. | | Hydrophilic head | The polar, water-soluble portion of a molecule. | | Hydrophobic tail | The non-polar, water-repelling portion of a molecule, often a lipid chain. | | Micelles | Aggregates of detergent molecules in an aqueous solution, formed above a certain concentration (critical micelle concentration), where the hydrophobic tails face inward and the hydrophilic heads face outward. | | Dialysis | A process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, used here to remove small molecules like detergents. | | Gel filtration chromatography | A chromatographic technique that separates molecules based on their size as they pass through a column packed with porous beads. | | Mass cut-off filter | A type of filter that allows molecules below a certain molecular weight (cut-off) to pass through while retaining larger molecules. | | Protein precipitation | A method used to separate proteins from a solution, often by adding organic solvents like acetone or by altering the pH or salt concentration. | | Anionic detergents | Detergents with negatively charged head groups (e.g., SDS), which can strongly disrupt membranes and protein interactions. | | SDS (Sodium Dodecyl Sulfate) | A common anionic detergent widely used in protein electrophoresis (SDS-PAGE) to denature proteins and impart a uniform negative charge. | | Non-ionic detergents | Detergents that do not carry a net electrical charge, making them generally milder and less denaturing than anionic detergents, often used to isolate membrane proteins while maintaining their activity. | | Zwitterionic detergents | Detergents that possess both a positive and a negative charge within the same molecule, resulting in a net neutral charge. They can disrupt protein-protein interactions without fully denaturing proteins. | | Isoelectric focusing (IEF) | A technique used to separate proteins based on their isoelectric point (pI), the pH at which a protein carries no net electrical charge. | | Ion exchange chromatography | A separation technique that relies on the reversible binding of charged molecules to an oppositely charged stationary phase. | | Enzymatic digestion | The process of breaking down molecules, in this context, cell walls, using specific enzymes. | | Lysozyme | An enzyme that catalyzes the degradation of bacterial cell walls by breaking the $\beta$-1,4-linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). | | NAM (N-acetylmuramic acid) | A component of the peptidoglycan layer in bacterial cell walls. | | NAG (N-acetylglucosamine) | A monosaccharide component of bacterial cell walls (peptidoglycan) and chitin. | | Peptidoglycan | A complex polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, providing structural support. | | Dounce homogenizer | A laboratory tool consisting of a pestle and a glass tube with a precise clearance, used for gentle cell lysis and homogenization with minimal heating. | | Shear stress | The force experienced by a material when it is subjected to external forces that cause deformation or rupture, used here to break cells. | | Viscous | Having a thick, sticky consistency due to internal friction. High viscosity in a solution can indicate successful cell lysis. | | Organelles | Membrane-bound structures within a eukaryotic cell that perform specific functions. Keeping them intact is sometimes desirable. | | Blender | A kitchen appliance adapted for laboratory use to homogenize or lyse samples through high-speed rotation of blades. | | Tissue chopper | A mechanical device used to cut tissues into very small, uniform pieces, facilitating subsequent extraction. | | Cryo-grinding | A method of grinding frozen samples in liquid nitrogen to make them brittle and easier to break down mechanically. | | Pestle and mortar | A set of kitchen tools used for grinding and crushing substances, adapted here for cryo-grinding solid samples. | | Bacteria | Single-celled microorganisms that lack a nucleus and other membrane-bound organelles. | | Yeast | Single-celled fungi. | | Fungi | A kingdom of organisms that includes yeasts, molds, and mushrooms. | | Plant cells | Eukaryotic cells that form plant tissues, characterized by a cell wall, chloroplasts, and a large central vacuole. | | Vial | A small container, typically made of glass or plastic, used for storing or transporting samples. | | High-speed shaking | The rapid agitation of a sample in a vial, used in bead beating to generate collisions that lyse cells. | | Mechanical stress | Physical forces applied to a sample that can cause it to deform or break. | | High frequencies | Referring to sound waves with a frequency above the range of human hearing, used in sonication. | | Vapor bubbles | Small pockets of gas that form and collapse in a liquid under the influence of acoustic waves (sonication), generating shock waves. | | Implode | To collapse or cause to collapse inward, referring to the rapid collapse of vapor bubbles in sonication. | | Shock waves | A wave of compression and rarefaction that travels through a medium; in sonication, these waves rupture cell membranes. | | Shear forces | Forces acting parallel to a surface, which can cause deformation or rupture. | | On ice | Maintaining samples at a low temperature (typically 0-4°C) to minimize degradation and enzymatic activity. | | Pulses | Short bursts of sonication, interspersed with cooling periods, to prevent overheating. | | Protein solubilization | The process of bringing proteins into solution, ensuring they are accessible for downstream analysis. | | Hydrophobic effect | The tendency of non-polar molecules or parts of molecules to aggregate in aqueous solution to minimize contact with water, which is a major driving force in protein folding and aggregation. | | Hydrophobic regions | Parts of a protein molecule that are non-polar and tend to associate with each other and avoid water. | | Hydrophilic heads | The polar, water-loving parts of molecules. | | Aggregates | Clumps or masses formed when molecules stick together, often due to hydrophobic interactions. | | Chaotropic ions | Ions that disrupt the hydrogen-bonding network of water, such as urea and guanidinium chloride. | | Urea | An organic compound with the formula $CO(NH_2)_2$, used as a chaotropic agent to denature proteins and enhance their solubility. | | Thio-urea | An organic compound with the formula $SC(NH_2)_2$, similar to urea but often more effective at disrupting hydrophobic interactions. | | Guanidinium chloride | A strong chaotropic agent with the formula $CH_5N_3 \cdot HCl$, used to denature proteins and increase their solubility. | | Disulfide bridges | Covalent bonds formed between the thiol groups (-SH) of two cysteine residues, which play a significant role in stabilizing protein structure. | | $\beta$-mercaptoethanol | A chemical compound with the formula $HSCH_2CH_2OH$, used as a reducing agent to break disulfide bonds in proteins. | | Dithiothreitol (DTT) | A powerful reducing agent with the formula $C_4H_{10}O_2S_2$, commonly used to cleave disulfide bonds in proteins. | | Trypsin | A serine protease enzyme that cleaves peptide bonds specifically after lysine (Lys) or arginine (Arg) residues, widely used in protein digestion for proteomics. | | Peptides | Short chains of amino acids linked by peptide bonds. | | Ionization | The process of converting neutral atoms or molecules into ions by adding or removing electrons. Essential for mass spectrometry analysis. | | LysC | A protease that cleaves peptide bonds specifically after lysine (Lys) residues, often used in combination with trypsin for more complete protein digestion. | | LC-MS (Liquid Chromatography-Mass Spectrometry) | A powerful analytical technique that combines the separation capabilities of liquid chromatography with the detection and identification capabilities of mass spectrometry. | | C18 solid phase extraction | A chromatographic technique using a stationary phase modified with an 18-carbon alkyl chain (C18), which is highly hydrophobic, to separate and purify peptides based on their hydrophobicity. | | Ion exchange chromatography | A separation technique that isolates molecules based on their charge. | | Peptide labelling buffer | A solution used to prepare peptides for specific labeling techniques, such as isotopic labeling, prior to analysis. | | Acetone precipitation | A method to precipitate proteins from a solution by adding cold acetone, which reduces protein solubility. | | SN (Supernatant) | The liquid remaining after a precipitate has been removed by centrifugation. | | RT (Room temperature) | The ambient temperature of a typical laboratory environment, usually around 20-25°C. | | Gel filtration | See gel filtration chromatography. | | Poreous beads | Small, permeable spheres used as the stationary phase in chromatography columns, allowing separation based on size. | | Buffer contaminants | Unwanted substances present in the buffer solution that can interfere with analysis. | | Spin tube format | A format where the chromatography column is integrated into a spin tube, allowing for rapid separation by centrifugation. | | Molecular weight cutoff filters | Filters designed to retain molecules above a specified molecular weight, used for size-based separation or concentration. | | SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) | A widely used technique for separating proteins based on their molecular weight. Proteins are denatured by SDS and migrate through a polyacrylamide gel matrix under an electric field. | | Stationary phase | The solid or liquid phase in chromatography that is fixed in place, over or through which the mobile phase moves. | | Hydrophobicity | The property of a substance that repels water; non-polar molecules are hydrophobic. | | Silica beads | Small spherical particles made of silicon dioxide, often used as a support material in chromatography. | | C-chains | Carbon chains, such as C18, attached to the stationary phase in chromatography to impart hydrophobicity. | | Hydrophobic interactions | Non-covalent attractive forces between non-polar molecules or regions in an aqueous environment, driven by the tendency to minimize contact with water. | | Organic solvent | A solvent that contains carbon, typically used in chromatography to dissolve non-polar compounds and elute them from a stationary phase. Examples include methanol and acetonitrile. | | Methanol | An organic alcohol ($CH_3OH$) commonly used as a solvent in chromatography. | | Acetonitrile | An organic solvent ($CH_3CN$) widely used in high-performance liquid chromatography (HPLC). | | Water-based buffer | An aqueous solution containing salts and/or other solutes used to maintain a specific pH and ionic strength, serving as a solvent in chromatography. | | Aqueous buffer | A buffer solution primarily composed of water. | | Eluate | The liquid that has passed through a chromatography column, containing the separated compounds. | | Concentrated target compound | The desired substance that has been purified and increased in concentration through a separation process. | | RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) | A type of HPLC that separates compounds based on their hydrophobicity. The stationary phase is non-polar, and the mobile phase is polar. | | High pH RP-HPLC | Reverse-phase HPLC performed at a high pH (alkaline conditions), which can alter the retention behavior of peptides compared to low pH RP-HPLC. | | SCX (Strong Cation Exchange) | A type of ion exchange chromatography that uses a stationary phase with negatively charged functional groups to bind positively charged molecules (cations). |