Sample preparation for bottom-up proteomics1 CG990 Methods in Proteomics Sample preparation for bottom-up proteomics Gabriela Lochmanová, Ph.D. Sample preparation for bottom-up proteomics2 Bottom-up proteomics • Peptides of suitable size for analysis by commonly available MS instrumentation • Identification and quantification of thousands of proteins from a single sample (without prior knowledge of the sample composition or reliance on antibodies) • Available open-source and commercially developed software tools • Possible to determine: ➢ peptide and inferred protein identity ➢ sites of post-translational modifications ➢ relative abundances of peptides among samples Sample preparation for bottom-up proteomics3 Bottom-up proteomics: Highlights • Starting material: different types of samples ➢ purified proteins ➢ cells ➢ tissues ➢ biological fluids developments in sample preparation methodologies and consumables • Trends in methodologies: ➢ Gel-free approaches - time-saving, ease, minimized sample loss ➢ Precipitation-free approaches – alternative methods used to remove detergents and other contaminants (suspension trapping, paramagnetic beads) traditional peptide cleanup methods using reverse-phase chromatography could be used for desalting but not detergent or polymer removal x new approaches that use coated magnetic particles enable removal of detergents, polymers, and salts pI MW Sample preparation for bottom-up proteomics4 Bottom-up proteomics − Basic pipeline • Series of steps to digest the protein into peptides through enzymatic digestion, followed by removal of contaminants before analysis by MS. • The choice of bottom-up workflow depends on the sample complexity and the goals of the experiment. Sample complexity − the number of proteins − dynamic range of protein concentration Homogenization / Lysis Protein quantification Reduction and Alkylation Enzymatic Digestion Sample Clean-up Fractionation Fractionation Sample Clean-up Peptide quantification Mass Spectrometry Sample preparation for bottom-up proteomics5 Homogenization / Lysis • Reagent-based methods • rapid, gentle, efficient, and reproducible • extraction of total protein or subcellular fractions • components non-compatible with MS need to be removed • suitable for cultured cells but may not be effective for some tissues • Physical disruption • lysis of a wide range of cells • high lysing efficiency • requires equipment • limited reproducibility • protein denaturation and aggregation can occur due to localized heating • cells disrupt at different times, so subcellular components may be subjected to ongoing disruptive forces Sample preparation for bottom-up proteomics6 Homogenization / Lysis Adaptive cavitation Technology ACT Ultrasonic cavitation − Quick changes of the pressure in a liquid sample during sonication − Formation of bubbles at local pressure decrease − Implosion of bubbles at critical size 5 microliters to 2 mililiters of sample (based on adaptor type) Sample preparation for bottom-up proteomics7 Bottom-up proteomics − Protein / Peptide quantification • Protein quantification − calculating how much enzyme (or chemical) is required for protein digestion • Peptide quantification − control of the yield of the protein cleavage process − for determining how much peptide sample should be injected for LC-MS/MS − essential in quantitative workflows in which equivalent amounts of total peptide are compared to reveal differences in relative abundance of individual peptides. • The colorimetric assays often interfere with substances used for tissue lysis such as detergents or disulfide-bond-reducing agents e.g., Bradford assay - not compatible with SDS; BCA assay - not compatible with DTT, β-ME, EDTA. • UV absorbance methods − Proteins contain three different aromatic amino acids carrying benzene, phenol, and indole rings, respectively. Each of these groups can be excited by UV light to fluoresce. Tryptophan Fluorescence − highly sensitive to its microenvironment with regard to proteins and to the polarity of the solvent. − quenched by several amino acids as well as many substances contained in buffers such as detergents − temperature and pH are influencing the intensity of Trp fluorescence Sample preparation for bottom-up proteomics8 Tryptophan Fluorescence (WF) assay • most detergents quench fluorescence at the concentration used for tissue lysis • Trp quantification – low interaction of detergents with the proteins in a buffer containing 8 M urea, Trp indole moieties freely exposed to the solvent; Emmax = 350 nm • suitable for high-complex samples but not for protein/peptide fractions Emission spectra of whole cell lysates (6, 12, and 18 μg of total protein) and pure tryptophan (0.05, 0.1, and 0.15 μg) in 2 mL of 8 M urea and 10 mM Tris-HCl, pH 7.8. Quenching effect of detergents and DTT. Reagent concentration refers to concentration in 2 μL of solution added to 2 mL of 8 M urea. Wisniewski et al., 2015, 87(8):4110-6 • working concentration of 0.05 - 25.0 mg/mL Sample preparation for bottom-up proteomics9 MicroBCA assay • colorimetric detection and quantitation of total protein / peptides • more suitable for low-complex protein / peptide samples compared to Trp assay • working concentration of 0.5-20.0 µg/mL • detergent-compatible • not compatible with reductants and chelatants (DTT, β-ME, EDTA…) Wisniewski et al., 2015, 87(8):4110-6 • the protein solution is mixed with an alkaline solution of cupric ions Cu2+ which chelate with the peptide bonds resulting in cuprous ions (Cu+). Purple-colored reaction product formed by the chelation of two molecules of BCA with one CuI+ ion exhibits absorbance at 562nm Sample preparation for bottom-up proteomics10 WF vs. MicroBCA assay Wisniewski et al., 2015, 87(8):4110-6 • both methods have similar sensitivity and reproducibility for protein determination in tissue lysate • WF assay appears to be more reproducible than the dye-based assay in the determination of peptide contents in protein digests Denaturation Reduction Alkylation 11 Reduction and Alkylation of proteins • Disulfide bonds of the proteins are irreversibly broken up and the optimal unfolding of the tertiary structure is obtained. • This chemical modification allows for proteins with a high number of disulfide bonds the successful identification as well as the highest peptide yield and sequence coverage. • Incomplete reduction and/or alkylation will impair the qualitative and quantitative results. • Undesired “over-alkylation” = the alkylation of nonthiol moieties: N-terminal amino acid>aspartic acid>glutamic acid>histidine>asparagine>lysine>tyrosine DTT− Dithiothreitol TCEP − tris-(2-carboxyethyl)-phosphine BME − β-mercaptoethanol R A Sample preparation for bottom-up proteomics IAA − iodoacetamide IAC − iodoacetic acid AA − acrylamide AA − chloroacetamide Protein with disulfide bridges Reduction cleaves disulfide bridges and allows unfolding Sample preparation for bottom-up proteomics12 Promega datasheet Reduction and Alkylation of proteins Sample preparation for bottom-up proteomics13 TPCK Trypsin: cleaves the carboxyl side of K and R; autolysis blocked SOLu-Trypsin: delivered in solution, stable at 4°C for one month SOLu-Trypsin Dimethylated: delivered in solution, stable at 4°C for one month; autolysis blocked Rapid Digestion Trypsin: digestion for 1 h at 70°C Platinum Trypsin: without non-specific proteolytic activity; autolysis-resistent LysC: cleaves the carboxyl side of K Glu-C (V-8 Protease): cleaves the carboxyl side of E (in ammonium bicarbonate and ammonium acetate buffers) cleavage can also occur at both E and D (in phosphate buffers) Asp-N: cleaves at amino side of D and cysteic acid residues (that result from the oxidization of C) Chymotrypsin: cleaves at the carboxyl side of aromatic acids - Y, F, W and L. Thermolysin: cleaves at the N-term of L, F, V, I, A, M at 65–85°C ProAlanase: cleaves the carboxyl side of P and A Enzymatic digestion of proteins Sample preparation for bottom-up proteomics14 Enzymatic digestion of proteins – alternative proteases Anal. Chem. 2020, 92, 9523−9527 Sample preparation for bottom-up proteomics15 Anal. Chem. 2020, 92, 9523−9527 Sequential digestion – trypsin - the protease of first choice in proteomics – specific studies can benefit from adding a sequential digestion step with trypsin – specific studies can benefit from usage of alternative protease Sample preparation for bottom-up proteomics16 Bottom-up proteomics − Pipeline for low-complex samples Purified protein HPLC fraction Subcellular fraction Aqueous solution MS-compatible solution Peptide quantification Protein quantification Reduction and Alkylation Enzymatic digestion Sample preparation for bottom-up proteomics17 Bottom-up proteomics − Pipeline for in-gel digestion Cells Tissues Biological fluids Solubilization buffer containing SDS/DTT Reduction and Alkylation Hydratation Enzymatic in-gel digestion Peptide extractionGel wash Dehydratation • 1-D or 2-D gel - visualization using an MS-compatible stain • The permeation of the enzyme to the gel is facilitated by the dehydration of the gel with acetonitrile and subsequent swelling in the digestion buffer (diffusion). • Smaller gel pieces - higher efficiency of the in-gel digestion • Relatively high enzyme concentration • Extraction of peptides by acidic extraction (50% acetonitrile/2.5% formic acid) in combination with sonication. • Advantage of the in-gel digestion: contaminants (e. g., detergents, salts) are already removed during electrophoresis • Disadvantage: limited effectiveness due to poor accessibility of the protease to proteins and/or inefficient release of peptides from the gel matrix Sample preparation for bottom-up proteomics18 Bottom-up proteomics − Pipeline for high-complex samples Cells Tissues Biological fluids Solubilization buffer containing detergents, chaotropic agents, salts Peptide quantification Enzymatic digestion Protein quantification Reduction and Alkylation Protein clean-up (MS-non-compatible component removal) Sample preparation for bottom-up proteomics19 Solubilization of high complex samples and Protein clean-up Homogenization in SDT buffer 4% SDS, 0.1M DTT, 0.1M Tris-HCl pH 7.6 Homogenization / Lysis • Clean-up and enzymatic digestion: Filter-Aided Sample Preparation (FASP) Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) Suspension Trapping (S-Trap) • Protein powder / Collected cells transfered to the vial with hot SDT buffer • Homogenization supported in Bioruptor, DNA fragmentation. • Complete protein solubilization ensured by incubation at 95°C, 2h. • Additional Clean-up: Ethylacetate extraction (EE) Critical Micelle Concentration (CMC) c < CMC - detergents occur as monomers c > CMC - detergent molecules organize in micelles which drive solubilization. Due to their sizes, the SDS and SDS mixed micelles cannot be separated from solubilized proteins by ultrafiltration. In FASP, concentrated urea enables contraction or dissociation of micelles. Sample preparation for bottom-up proteomics20 Impact of SDS in proteomic approaches • Suppresses enzyme activity during protein digestion • Affects reversed-phase LC and its surface activity − SDS impairs chromatographic separation of peptides (shift in retention time) • Suppresses ionionization of other species during electrospray ionization (mass spectra of SDS dominates due to its ready ionization ability and high-abundance, signals of SDS-peptide adducts are formed) • Facilitates protein solubilization • SDS removal (protein precipitation, column based approaches, dialysis, …). • SDS-assisted protein digestion has been shown to enhance the detection of membrane proteins Possible solutions: • MS-compatible alternatives to SDS (e.g., cleavable surfactants: ProteaseMAX, RapiGest, PPS Silent Surfactant, octyl β-D-glucopyranoside, n-dodecyl β-D-maltoside, and digitonin) Sample preparation for bottom-up proteomics21 Protein Clean-up: Filter-Aided Sample Preparation (FASP) MWCO molecular weight cut-off The molecule of a given molecular weight (Da) retained with 90% efficiency by the membrane ultrafilter 8M urea IAA/8M urea urea SDS DTT Proteins in SDT buffer Centrifugation Ammonium bicarbonate urea IAA Centrifugation Trypsin Centrifugation Nucleic acids Proteins High MW material Incubation Centrifugation Purified peptides Nature Methods 6, 359 - 362 (2009) • Excellent performance for samples between 25 and 100 μg of total protein Sample preparation for bottom-up proteomics22 • multienzyme digestion (MED) FASP (Anal. Chem. 2012, 84, 2631−2637) − sequential digestion of protein material with a second or third enzyme − increased number of identified proteins and their sequence coverage − increased depth of identification of phosphorylation sites Anal. Chem. 2012, 84, 2631−2637 Protein Clean-up: Modifications of FASP Sample preparation for bottom-up proteomics23 • enhanced FASP (eFASP) (J. Proteome Res. 2014, 13, 1885 − 1895) − pre-passivation of Microcon filter surfaces with 5% TWEEN-20 to enhance peptide recovery and uses a surfactant (0.2% deoxycholic) during detergent steps and digestion to increase trypsin efficiency • 96-well format for high-throughput processing − plates with a 10 MWCO membrane; disadvantage: low liquid transfer speeds during centrifugation (Proteomics 2013, 13, 2980–2983) − MStern-blot (MStern) - plates with large-pore hydrophobic polyvinylidene fluoride (PVDF) membrane which efficiently adsorbs proteins; fast liquid transfer through the membrane using a vacuum manifold. (Mol Cel Proteomics 2015 Oct;14(10):2814-23) − polyethersulfone (PES) filtration membrane enables to use 10% isopropanol (IPA) as a wetting agent, resulting in a reduction of 50% in the time required for buffer exchange. IPA reduces surface tension between the aqueous layer and the membrane. Reduced critical micelle concentrations of detergents due to presence of alcohol is balanced by urea. (PLoS ONE 2017, 12(7): e0175967) J. Proteome Res. 2014, 13, 1885 − 1895 Mol Cel Proteomics 2015 Oct;14(10):2814-23 Protein Clean-up: Modifications of FASP Sample preparation for bottom-up proteomics24 Peptide Clean-up: Ethylacetate extraction (EE) Curr Protoc Protein Sci. 2010 February ; CHAPTER: Unit–16.12. density (g/mL) EE 0.902 water 0.998 • Ethyl acetate − highly volatile − low-solubile in water − efficient solvent for several detergents (octylglucoside, SDS, Triton X-100, NP-40….) Water-saturated EE • EE extraction − two-way process : partition of hydrophobic molecules to organic solvent from the aqueous solution, and partition of hydrophilic molecules in the organic solvent to the aqueous phase − extraction solvent of highest quality has to be used − acid washed glass bottles and pipettes should be used for the storage of EE − poly-propylene or poly-ethylene tubes and pipette tips can be used for short term extraction − five to ten times the volume of solvent to peptide solution in each extraction − loss of particular peptides (e.g., larger peptides) Vortex Centrifugation Upper layer removal Peptides with traces of SDS Purified peptides EE SDS Sample preparation for bottom-up proteomics25 Alternative methods for digestion / clean-up Protein Aggregation Capture (PAC) on microparticles of various surface chemistry – a mechanism which uses the phenomenon of non-specifically immobilizing precipitated and aggregated proteins on any type of sub-micron particles irrespective of their surface chemistry. (Mol Cell Proteomics 18: 1027–1035, 2019) Nature protocols JANUARY 2019 | 68 – 85 EtOH 50% EtOH 80% Trypsin/AB Beads clumping (Mol Cell Proteomics 18: 1027–1035, 2019) Sample preparation for bottom-up proteomics26 Alternative methods for digestion / clean-up Single-Pot Solid-Phase-enhanced Sample-Preparation (SP3) – a paramagnetic bead–based approach – uses PAC mechanism for exchange or removal of contaminants (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) – non-selective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads – elution of purified material in aqueous conditions (Nature protocols JANUARY 2019 | 68 – 85) PROTEIN LYSATE 0.1 mg - 500 mg CONTAMINANTS Detergents 0-20% Chaotropes 0-8M Salts 0-1M Solvents 0-50% ELUTED PEPTIDES • polystyrene core made by a free radical emulsion polymerization of styrene and acid monomer • layer of magnetite • carboxylated polymer surface surface is modified to minimize non-specific binding of proteins SeraMag beads Sample preparation for bottom-up proteomics27 SP3 vs. FASP Front plant Sci 2021 Mar 10;12:635550 Sample preparation for bottom-up proteomics28 Peptide Clean-up: SP2 J Proteome Res. 2019 April 05; 18(4): 1644–1656. • RP-LC C18 resin − effective for removing salts and concentrating peptides − available in a wide variety of easy-to-use formats (e.g., Stage-Tips, Sep-Pak Cartridges, Micro SpinColumns) − however, C18 concentrates polymeric species such as polyethylene glycol (PEG) and common detergents (e.g., NP-40, SDS, Triton X). SP2 − lower binding capacity for peptides than for proteins: 50 and 200 ng/ μg for simple and complex peptide mixtures X 100 μg of protein/μg of particle − suitable for variety of contaminants; (not suitable for Tris removal) − peptides characterized as long, hydrophobic, or highly negative are more reproducibly processed with SP2 than with C18 Sample preparation for bottom-up proteomics29 Magnetic racks Sample preparation for bottom-up proteomics30 Alternative methods for digestion / clean-up Suspension trapping (STrap) – an instant creation of a fine protein particulate suspension from an SDS-solubilized protein solution, which can then be trapped by the filter – aggregation of the suspension minimized by adding the protein–SDS mixture to an ethanolic solution at a near-neutral pH – SDS monomers are soluble in ethanolic solution and are filtered out together with other contaminants • sample lysis and solubilization in 5% SDS • protein denaturation by acidification to pH < 1 and subsequent exposure to a high concentration of ethanol • such three-stage denaturation ensures complete destruction of undesired enzymatic activity such as proteases and phosphatases • reduction and alkylation can be performed in 5% SDS, precluding precipitation, or alternatively on-column after the denatured proteins are trapped • denatured, non-digested proteins are bound to the S-Trap via centrifugation or vacuum • multiple weak-affinity interactions hold the undigested protein within the pores of the derivatized silica S-Trap • captured proteins are presented with maximal surface area allowing them to be washed fully free of all contaminants in only minutes: detergents, PEG, glycerol, detergents, salts, Laemmli loading buffer, etc. Sample preparation for bottom-up proteomics31 Solubilization of high complex sample for PTM enrichment Homogenization in Urea buffer 9M Urea, 20mM HEPES pH 8.0 Clean-up: reversed-phase Solid-Phase Extraction (SPE; Sep-Pak® C18 Cartridges) The hydrogen bond interaction between urea and the peptide groups opens the entrance for water, and contributes to the unfolding denaturation of protein. Zhang et al., 2017, Phys. Chem. Chem. Phys., 19, 32007-32015 10mL Urea / HEPES Cell count ~1×10E8 PBS wash Trypsin Enzymatic digestion Sonication Dilution (final concentration of urea 2M)Centrifugation Elution: 0.1%TFA/40% ACN Digest acidification: pH ˂3 Purified peptides Reduction and Alkylation Lyophilization (TFA removal) Sample preparation for bottom-up proteomics32 PTM enrichment lyphilized peptides IAP buffer ImmunoAffinity Purification (IAP) pH~7.0 Anti-PTM Ab Incubation 2h, 4°C Centrifugation Wash Elution 0.15% TFA Centrifugation Wash Clean-up C18 SpinTip PTMScan® Technology (Cell Signaling Technology) - peptide enrichment by immunoprecipitation using a specific bead-conjugated antibody in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS/MS) for quantitative profiling of post-translational modification (PTM) sites in cellular proteins. phosphorylation (PhosphoScan®) ubiquitination (UbiScan®) acetylation (AcetylScan®) methylation (MethylScan®) protein A agarose beads 10 - 20 mg peptides Pept conc measurement microBCA assay Sample preparation for bottom-up proteomics33 Phosphoproteomics − predominately employs bottom-up mass spectrometry (MS) based techniques − phosphorylation occurs at single (mono-) or multiple (multi-) sites and can co-occur with other PTM types to generate different „proteoforms“ − phospho-serine/threonine (pSer/pThr) sites using MS techniques has improved, but the determination of tyrosine (pTyr) sites is challenging because the abundance of pTyr is significantly lower than that of pSer/pThr − phosphopeptides tend to have low ionization efficiency due to (i) phosphate groups tending to lose protons to carry negative charges (ii) background presence of large amounts of unphosphorylated peptides − phosphopeptides are low abundant relative to non-phosphorylated counterparts Analytica Chimica Acta 1129 (2020) 158e180 Sample preparation for bottom-up proteomics34 Phosphoproteomics Affinity-based phosphopeptide enrichment − selectively binds the negatively charged phosphate groups of the p-peptide to metal ions or metal oxide or employs Ab − Elution from IMAC and MOAC by displacing the negatively charged phosphate with a basic buffer − IMAC result in higher detection of multi-p-peptides, while TiO2 enrichment results in a high identification number of mono-p-peptides due to dissociation difficulty (incomplete elution) Analytica Chimica Acta 1129 (2020) 158e180 Fe3+ Ga3+ TiO2 ZrO2 In2O3 − TiO2-based approaches: higher selectivity and specificity, robustness, amphoteric ion-exchange characteristics, tolerance towards many reagents (stable in wide pH ranges) − Different configurations for operating MOAC-TiO2: spin columns, analytical columns, miniaturized columns, nanoparticles, magnetic beads, … − pTyr a smaller fraction of the p-proteome; anti-Tyr Ab used for selective enrichment (poor reproducibility, low sensitivity, limited availability/ variability of Ab, limited availibility of bulk starting materials, high costs) Sample preparation for bottom-up proteomics35 Phosphoenrichment High-Select ™ TiO2 Phosphopeptide Enrichment Kit – spherical porous TiO2, optimized buffers, spin columns – provide enhanced enrichment and identification of phosphopeptides with minimal nonspecific binding – phosphopeptide yields are typically ~1-3% of the starting sample – starting material: lyophilized peptide samples free of detergents and salts pH>10 0.5 – 3.0 mg peptides solubilized in lactic acid Centrifugation Wash Equilibrated spin column spin column with bound phosphopeptides Elution NH4OH pH<3 Elution Buffer evaporation Acidification Sample preparation for bottom-up proteomics36 Enzymatic digestion 10 20 30 40 50 MGKKQNKKKV EEVLEEEEEE YVVEKVLDRR VVKGKVEYLL KWKGFSDEDN 60 70 80 90 100 TWEPEENLDC PDLIAEFLQS QKTAHETDKS EGGKRKADSD SEDKGEESKP 110 120 130 140 150 KKKKEESEKP RGFARGLEPE RIIGATDSSG ELMFLMKWKN SDEADLVPAK 160 170 180 EANVKCPQVV ISFYEERLTW HSYPSEDDDK KDDKN 10 20 30 40 50 MGKKQNKKKV EEVLEEEEEE YVVEKVLDRR VVKGKVEYLL KWKGFSDEDN 60 70 80 90 100 TWEPEENLDC PDLIAEFLQS QKTAHETDKS EGGKRKADSD SEDKGEESKP 110 120 130 140 150 KKKKEESEKP RGFARGLEPE RIIGATDSSG ELMFLMKWKN SDEADLVPAK 160 170 180 EANVKCPQVV ISFYEERLTW HSYPSEDDDK KDDKN 10 20 30 40 50 MGKKQNKKKV EEVLEEEEEE YVVEKVLDRR VVKGKVEYLL KWKGFSDEDN 60 70 80 90 100 TWEPEENLDC PDLIAEFLQS QKTAHETDKS EGGKRKADSD SEDKGEESKP 110 120 130 140 150 KKKKEESEKP RGFARGLEPE RIIGATDSSG ELMFLMKWKN SDEADLVPAK 160 170 180 EANVKCPQVV ISFYEERLTW HSYPSEDDDK KDDKN Characterization of enriched (low-abundant) proteins bait Identification / Quantification of • PTMs on bait protein • Interacting protein partners 10 20 30 40 50 MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL 60 70 80 90 100 IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG Sample preparation for bottom-up proteomics37 variant I variant II Characterization of enriched sequence variants variant I variant II variant I variant II Enzymatic digestion Enzymatic digestion Sample preparation for bottom-up proteomics38 Practical course C8302 III. Phosphoenrichment II. SP3-based digestion / clean-up I. In-gel digestion Sample preparation for bottom-up proteomics39 Particular figures were created with BioRender.com. Thank you for your attention! Gabriela Lochmanová gabriela.lochmanova@ceitec.muni.cz