Characterization of Intact Proteins by ESI-LC/MS

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1 Characterization of Intact Proteins by ESI-LC/MS 1

2 Characterization of Intact Proteins Recombinant Proteins integrity of construct extent t and ID of post-translational t ti l or other modifications Native Proteins Protein-Protein or Protein-Small Molecule Interactions Top-down sequencing 2

3 Challenges Concentration usually need pmols of relatively e pure protein Sample Handling buffer components Problematic classes of proteins membrane proteins; very large proteins; highly folded, disulfide rich proteins Other problems aggregation; precipitation it ti 3

4 Typical Biological Buffer Components Buffers: HEPES, Tris, TBS, PBS Detergents: SDS, Triton, NP-4, CHAPS (see Appendix) Chaotropes: guanidine, urea Reductants: DTT, 2-mercaptoethanol, ascorbic acid Salts: NaCl, KCl Chelators: EDTA Protease Inhibitors: aprotinin, leupeptin, PMSF, pepstatin Stabilizers: glycerol, mannitol, PEG Bacteriostats: NaN 3 4

5 Electrospray vs MALDI Electrospray direct coupling to LC for on-line desalting/preconcentration multiple charging allows analysis of proteins with MW s greater than the mass range of the mass spectrometer ete resolution of species with relatively small molecular weight differences (methylation, oxidation, etc.) soft ionization i (preserve some noncovalent interactions) MALDI off-line sample prep relatively low resolution protein spectra more tolerant of salts than ESI ESI/MALDI often complementary 5

6 Useful Conversions 1 mm = 1 µm 1 µm = 1 pmol/µl 1 mg/ml = 1 µg/µl To convert mg/ml or µg/µl / Lto pmol/µl: pmol/µl = (mg/ml x 1 6 )/protein MW Example: 1 mg/ml of a 3 kda protein [(1 mg/ml) x 1 6 )/3,] = 33.3 µm 6

7 Electrospray Produces Multiply Charged Ions In + ion mode, 1 s to 1 s of charges via protonation or adduct formation (Na+, K+) Parameters for optimization include: cone/skimmer voltages (charge stripping) heated capillary temperature desolvation gas flow buffer composition (for noncovalent interactions) ti Usually use volatile buffers (NH 4 HCO 3 ) in the ph range to preserve interactions 7

8 ESI of Myoglobin (MW ~ 17 kda) myo cal q3t27 37 (7.32) Sm (Mn, 4x8.); Cm (363:391) myo cal q3t27 37 (7.32) Sm (Mn, 4x8.); Cm (363:391) TOF MS ES+ 518 TOF MS ES % m/z % m/z

9 Calculating Protein MW s from Adjacent Charge Statest M = molecular weight of protein z = charge state Y X = (M+z)/z Y = (M+z+1)/(z+1) In ntensity m/z X To calculate the charge state (z) of X, z x =(Y1)/(X (Y-1)/(X-Y) Y) To calculate the molecular weight of the protein, M = (X * z x ) z x = (Y * z y ) - z y 9

10 X = Calculating Protein MW s: Myoglobin Example Y = z x = (Y-1)/(X-Y) = ( )/( ) = , X = 17 and Y = 18 M = (X * z x x) z x = * = Predicted MW of Myoglobin = If calculated for multiple adjacent charge states, an average MW and standard deviation may be determined. ***Must be sure X and Y are from same series!!!!! 1

11 Calculating Protein MW s: Automated t Calculation l myo cal q3t27 37 (7.32) AM (Cen,8, 8., Ht,5.,.,1.); Sm (Mn, 4x8.); Cm (363:391) A18 1 A A A TOF MS ES+ 584 A: ±.2 B: ±.19 A A A A % A A B A B A B A B B A B m/z

12 Calculating Protein MW s: Deconvoluting from m/z to MW myo cal q3t27 37 (7.32) Sm (Mn, 2x3.); Cm (361:396) TOF MS ES+ 675 myo cal q3t27 37 (7.32) M1 [Ev ,It12] (Gs,.75,724:2357,1.,L3,R1); Sm (Mn, 2x e4 % MaxEnt1 Measured: Deconvolution Predicted: Error = 1.5 Da = % 9% % m/z mass 12

13 Deconvoluting from m/z to MW: Artifacts from Deconvolution myo cal q3t27 37 (7.32) M1 [Ev-22263,It5] (Gs,.75,716:2461,2.,L3,R1); Sm (Mn, 2x3.); Cm (36:391) TOF MS ES+ 6.77e3 Myoglobin Artifact peaks at 1/2X, 2X, 3X, etc. % Look back at raw data to rule out artifacts mass

14 Deconvoluting from m/z to MW: Artifacts can be identified by examining raw data myo cal q3t27 37 (7.32) AM (Cen,8, 8., Ht,5.,.,1.); Sm (Mn, 4x8.); Cm (363:391) A18 1 A A A Arrows show where ions should be for a protein at MW 3396 TOF MS ES+ 584 A: ±.2 B: ±.19 A A A A % A A B A B A B A B B A B m/z

15 Deconvoluting from m/z to MW: Choosing the m/z range CDK 4 lot Holmes. B q3t (8.696) Sm (Mn, 3x5.); Cm (26:276) CDK 4 lot Holmes. B TOF MS ES+ q3t (8.696) M1 [Ev4178,It2] (Gs,.75,3:25,2.,L3,R5); Sm (Mn, 3x5.); C MaxEnt % % m/z mass

16 Deconvoluting from m/z to MW: Choosing the m/z range CDK 4 lot Holmes. B q3t (8.696) Sm (Mn, 3x5.); Cm (26:276) CDK 4 lot Holmes. B TOF MS ES+ q3t (8.696) M1 [Ev-17412,It2] (Gs,.75,1642:249,2.,L4,R1); Sm (Mn, 3x diphos monophos FL nonphos % % m/z mass

17 Sample Handling/Cleanup for Sample Handling ESI store samples at -2 to -8 o C keep on ice when in use, use refrigerated autosampler avoid multiple freeze/thaw cycles (aliquot) Dilution if protein concentration sufficiently high (mg/ml quantities) dilute with 5/5 MeOH/water, 1-5% formic or acetic acid Infuse at 1-5 µl/min Reversed-phase C2, C4, C8, C18, Poros and other polymeric resins perfusive resins (Poros), larger particle sizes, or short columns can allow for higher flow rates for rapid desalting small particle size, silica based supports offer optimal chromatographic resolution extremely hydrophobic proteins may be difficult to elute from more hydrophobic supports 17

18 Sample Cleanup for ESI Reversed-phase formats pipette tip desalting homemade, ZipTips (Millipore), step elution Cartridges Dionex, Waters, Michrom can gradient elute with an LC or step elute/collect for infusion For both tips and cartridges off-line, wet packing with organic prior to use equilibrate with.1% TFA or.1% formic acid load sample slowly; dilute sample if organic is > 1% in sample wash extensively with.1% formic acid; may wash with 1% MeOH or MeCN to assist in removal of hydrophobic buffer components elute with 8% MeOH or MeCN containing.1-1% 1% formic acid 18

19 Homemade Poros RP Perfusion Column Upchurch ZDV PEEK Union (P742) Stainless Frit Upchurch (C47) PEEK Sleeve From LC Fused Silica Blue 25µm id PEEK Tubing (15-2 cm) Poros 1R1, 1R2, or 1R3 Load at 5-1 µl/min Elute at 2-3 µl/min To MS 19

20 Sample Cleanup for ESI Ultrafiltration molecular weight cut off (MWCO) spin cartridges (Amicon, Centricon, etc.) low MW species pass through membrane; species of MW > membrane cutoff do not pass through MWCO ranges: 3, 1, 3, 5, and 1 kda choose a MWCO ~ 1/2 the MW of the protein to be retained can be used for salt and detergent removal detergents will not pass through the membrane if concentration > CMC for small protein amounts, use with caution irreversible adsorption to the membrane Size Exclusion spin columns (BioRad, etc.) higher MW components elute first, low MW components retained 2

21 Sample Cleanup for ESI Precipitation for removal of detergents and other components not compatible with mass spec or protein digestion Methods (See appendix for protocols) chloroform/methanol precipitation acetone ethanol Resolubilization may be a problem. Try the following: 5% MeOH, 5% formic acid may initially add a few ml of 7% formic acid, diluting quickly to < 1% (avoid formylation of protein) 6M Guanidine or 4M Urea may use a few ml of hexafluoroisopropanol or hexafluoroacetone (highly toxic, use only in hood, vent MS source) Requires a lot of protein (1s to 1s of µg s) 21

22 Sample Cleanup for ESI Ion Exchange Anion Exchange removal of SDS homemade pipet tips/columns, commercial cartridges acidify sample to ~ph 2-3; pass sample slow over anion exchanger protein passes through, negatively charged SDS binds Cation Exchange select cation exchange compatible ph, elution conditions; prep sample for compatibility with CX Hydrophilic Interaction bind in high h organic; elute in high h aqueous can be used for detergent removal may be coupled on-line with MS for analysis of membrane or other hydrophobic proteins 22

23 Microdialysis Sample Cleanup for ESI Pierce Slide-A-Lyzer Mini; Amika Microdialyzer Affinity it Capture Ni 2+ chelate for His-Tag; Fe 3+ /Ga 3+ chelate for phosphoproteins; monomeric avidin for biotin-tagged proteins; antibody binding Prep for noncovalent interaction studies Must use a buffer which preserves native conformation/ interactions Typically, 1-5mM ammonium acetate, ph May have to buffer exchange prior to MS 23

24 Production of Recombinant Proteins Point mutations, fusion protein sequences, linker sequences, Buffers and other additives, i degradation, aggregation Post-translational modification 24

25 Commonly Used Expression Systems 25

26 Common Recombinant Protein Purification Strategies t 1. GST Fusion Proteins GST Sequence (~ 26 kd) Protein of Interest Sequence -COOH Linker Sequence (May contain a protease site, i.e., thrombin, TEV protease) -Purify using an immobilized glutathione column 2. His-tagged Proteins His Tag MSYYHHHHHHXXXX Protein of Interest Sequence -COOH -Purify using an immobilized Ni 2+ column. Tag may be N-terminal or C-terminal 26

27 Common Observations in Protein MS Loss of N-terminal Methionine (-131 Da) N-terminal Acetylation (+42 Da) Phosphorylation (+8 Da) Glycosylation (heterogeneous, variable) Degradation/Truncation (N- or C-terminal) Glutathionylation of GST Fusions (+35) Phosphogluconoylation (His-tag in E. coli, or 258) Mutation Combinations of the above Other 27

28 Phosphorylation (+8 Da) CFM-S T9 Hassell, A q3t (8.482) Sm (Mn, 3x5.); Cm (437:451) CFM-S T9 Hassell, A TOF MS ES+ q3t (8.482) M1 [Ev-65849,It9] (Gs,.75,1378:1898,2.,L4,R2); Sm (Mn, 3x5.); CFM-S T9 Hassell, A q3t (8.482) Sm (Mn, 3x5.); Cm (437:451) TOF MS ES % % % m/z MaxEnt Multiple Phosphorylation States m/z mass

29 Biotinylation (mass shift varies) FtzF1 LBD + NHS-LC-biotin Consler, T FtzF1 LBD + NHS-LC-biotin Consler, T q3t25 43 (7.641) Sm (Mn, 3x5.); Cm (396:431) TOF MS ES+ q3t25 43 (7.641) M1 [Ev-53519,It2] (Gs,.75,927:2495,2.,L3,R1); 2 Sm (Mn, 3x5.); e % MaxEnt1 % nd Series: modified His-Tag m/z mass

30 Ni Adduct Formation After His-Tag Purification Untreated WR AKT Fin GDE LCT (6.273) M1 [Ev354,It2] (Gs,.5,131:1551,2.,L33,R1); Sm (Mn, 2x e3 Treated 6M Gu, 1mM EDTA, 2M DTT, 6 o C Column Temp WR AKT LCT (5.98) M1 [Ev-13181,It2] (Gs,.5,972:1237,2.,L33,R1); Sm (Mn, 2x e4 FL Protein FL Protein % +~59 Da (Ni?) Phos % Phos mass mass

31 Reasons aggregation Proteins that don t fly! tightly folded/extensive disulfide network size other Approach denature chaotrope/reductant (add 8M guanidine, 2M DTT, 37 o C temperature (6 o C) 31

32 PB Erb4999 no tr 6C LCT (8.547) Sm (Mn, 3x5.); Cm (453:59) % Proteins that don t fly! Standard Conditions TOF MS ES LCT (8.712) Sm (Mn, 3x5.); Cm (456:515) TOF MS ES+ 32 Treated with 6M Guanidine % LCT (6.163) Sm (Mn, 3x5.); Cm (112:141) Column Temp = 6 o C TOF MS ES+ 289 % m/z

33 Keys to the Analysis of Large Proteins Utilize a TOF mass analyzer Mass Resolution > 1, Internal mass correction Lock spray or dual spray Good chromatography Separate protein from buffer salts, modifiers, and other smaller proteins 33

34 hfas Theoretical MW = 274,629 Da 8 x TIC Scan FAS_h5.d Counts vs. Acquisition Time (min) Measured MW = 274,631 lock mass 5 ug (2pmol) of total protein injected Courtesy of Jon Williams, GSK 34

35 rfas Theortical MW = 273,852 Da Measured MW = 273,854 lock mass ~7 ug (2pmol) of total protein injected Courtesy of Jon Williams, GSK 35

36 Common Reasons for MW Discrepancies 1. Submitter supplies sequence with error(s) () 2. Post-translational modifications 3. Mutations to protein 4. Wrong protein 36

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