Characterization of Intact Proteins by ESI-LC/MS

Characterization of Intact Proteins by ESI-LC/MS 1

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

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

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

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

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

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 7-7.5 range to preserve interactions 7

ESI of Myoglobin (MW ~ 17 kda) myo cal q3t27 37 (7.32) Sm (Mn, 4x8.); Cm (363:391) 1 942.8212 998.238 893.2551 myo cal q3t27 37 (7.32) Sm (Mn, 4x8.); Cm (363:391) 16.5437 1 16.54 TOF MS ES+ 518 TOF MS ES+ 585 16.5437 % 848.6454 1131.1853 163.86 163.8629 167.16 167.165 1211.992 156 157 158 159 16 161 162 163 164 165 166 167 168 169 17 171 172 173 174 175 1884.5973 m/z % 88.283 135.61 1413.762 1696.2451 212.256 1542.1244 771.61 189.4971 2126.6787 139.1267 1418.178 1422.5162 1546.9144 171.5554 1896.371 2422.725 2133.344 243.3567 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 8 m/z

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

X = 998.23 Calculating Protein MW s: Myoglobin Example Y = 942.82 z x = (Y-1)/(X-Y) = (942.82-1)/(998.23-942.82) = 16.997, X = 17 and Y = 18 M = (X * z x x) z x = 998.23 * 17-17 = 16952.91 Predicted MW of Myoglobin = 16951.5 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

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 A17 942.8455 998.2512 A19 893.2781 A16 16.578 TOF MS ES+ 584 A: 16952.96±.2 B: 175.74±.19 A2 848.6612 A15 1131.2175 A14 1211.9413 A9 1884.634 % A21 88.2936 A13 135.819 B13 139.1353 A12 1413.7439 B12 1418.147 A11 1542.1663 B11 1546.9542 A1 1696.2798 B1 171.568 1313.245 1422.5557 1551.865 176.9254 1321.4646 1431.39273927 1694.668668 1718.8639 B9 189.529 1896.4167 A8 212.789 192.3551 2117.8416 B8 2126.71 2133.3728 214.166 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 11 m/z

Calculating Protein MW s: Deconvoluting from m/z to MW myo cal q3t27 37 (7.32) Sm (Mn, 2x3.); Cm (361:396) 998.22 1 942.82 893.27 16.53 TOF MS ES+ 675 myo cal q3t27 37 (7.32) M1 [Ev-151748,It12] (Gs,.75,724:2357,1.,L3,R1); Sm (Mn, 2x3 16953. 1 4.88e4 % 1131.2 1211.94 135.6 MaxEnt1 Measured: 16953 Deconvolution Predicted: 16951.5 Error = 1.5 Da = 1884.4747.9% 9% % 1413.74 1696.25 212.3 1542.9 171.49 189.41 2126.77 176. 1896.32 2133.23 16934. 1758. 17951. 8 1 12 14 16 18 2 22 m/z 15 16 17 18 19 mass 12

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) 16952. 1 TOF MS ES+ 6.77e3 Myoglobin Artifact peaks at 1/2X, 2X, 3X, etc. % Look back at raw data to rule out artifacts. 8476. 176. 3396. 852. 9418. 1132. 13186. 157. 1758. 2543. 2264. 1972. 21192. 23734. 28254. 29668. 2637. 3412. 8 1 12 14 16 18 2 22 24 26 28 3 32 34 13 mass

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 A17 942.8455 998.2512 A19 893.2781 A16 16.578 Arrows show where ions should be for a protein at MW 3396 TOF MS ES+ 584 A: 16952.96±.2 B: 175.74±.19 A2 848.6612 A15 1131.2175 A14 1211.9413 A9 1884.634 % A21 88.2936 A13 135.819 B13 139.1353 A12 1413.7439 B12 1418.147 A11 1542.1663 B11 1546.9542 A1 1696.2798 B1 171.568 1313.245 1422.5557 1551.865 176.9254 1321.4646 1431.39273927 1694.668668 1718.8639 B9 189.529 1896.4167 A8 212.789 192.3551 2117.8416 B8 2126.71 2133.3728 214.166 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 14 m/z

Deconvoluting from m/z to MW: Choosing the m/z range CDK 4 lot 18477-1 Holmes. B q3t283 267 (8.696) Sm (Mn, 3x5.); Cm (26:276) 814.39 1 892.91 893.41 CDK 4 lot 18477-1 Holmes. B TOF MS ES+ q3t283 267 (8.696) M1 [Ev4178,It2] (Gs,.75,3:25,2.,L3,R5); Sm (Mn, 3x5.); C 179 1 47.2 814.5 776.69 776.38 1164.53 11.3 1221.7 1246.91 MaxEnt1 1385.34 % % 15 5 75 1 125 15 175 2 225 m/z 725 7275 73 7325 735 7375 74 7425 mass

Deconvoluting from m/z to MW: Choosing the m/z range CDK 4 lot 18477-1 Holmes. B q3t283 267 (8.696) Sm (Mn, 3x5.); Cm (26:276) 1666.16 1 CDK 4 lot 18477-1 Holmes. B TOF MS ES+ q3t283 267 (8.696) M1 [Ev-17412,It2] (Gs,.75,1642:249,2.,L4,R1); Sm (Mn, 3x5. 23.9 73266. 1 982 diphos monophos 73186. 174.82 1745.47 1785.93 FL nonphos 1832.79 % 1879.59 % 7316. 733. 73346. 1929.1 1981.21 236.17 292.6 294.28 2153.71 2221.12 229.36 2361.88 2443.21 725. 7292. 72884. 72772. 72528. 73374. 73432. 735. 746. 73672. 73828. 7417. 74384. 2445.1 17 18 19 2 21 22 23 24 m/z 725 7275 73 7325 735 7375 74 7425 16 mass

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

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

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

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

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

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

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 7.-7.5 May have to buffer exchange prior to MS 23

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

Commonly Used Expression Systems 25

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

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, + 178 or 258) Mutation Combinations of the above Other 27

Phosphorylation (+8 Da) CFM-S T9 Hassell, A q3t134 443 (8.482) Sm (Mn, 3x5.); Cm (437:451) 148.64 1 917.65 CFM-S T9 Hassell, A TOF MS ES+ q3t134 443 (8.482) M1 [Ev-65849,It9] (Gs,.75,1378:1898,2.,L4,R2); Sm (Mn, 3x5.); 1 1 37526. 3766. 37688. 95.2 37368. CFM-S T9 Hassell, A q3t134 443 (8.482) Sm (Mn, 3x5.); Cm (437:451) 171.37 1 1699.38 1714.3 TOF MS ES+ 8.65 37448. 37768. 1313.98 1717.81 37288. % 1695.76 1718.82 % 893.53 1687.46 167.3 1675.5 1686.4 172.64 1723.58 1731.14 1738.721742.53 % 3728. 873.51 584.31 78.44 752.45 58.36 553.39 1332.81 139.9 1443.4 1447.51 167 168 169 17 171 172 173 174 m/z MaxEnt1 37166. 1 2 3 4 5 6 155.32 Multiple Phosphorylation States 5 75 1 125 15 175 2 225 m/z 37 372 374 376 378 28 mass

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.); 1494.36 1 11 3136. 1 1.43e4 1426.5 1569.3 1364.52 1651.56 3122. +4 % 1349.8 137.71 1293.58 1255.46 1241.86 1194.13 1162.56 1149.96 1743.25 1762.13 1865.7 1982.29 MaxEnt1 % +2 3682. +3 317. +5 2nd Series: modified His-Tag +6 324. 1137.42 182.41 291.74 2114.31 2241.4 2265.21 2413.3 2439.37 +2 +3 +4 31198. +1 3154. +7 386. 3342. 32378. 3. 3522. 32216. 32724. 32786. 1 12 14 16 18 2 22 24 m/z 3 35 31 315 32 325 29 mass

Ni Adduct Formation After His-Tag Purification Untreated WR AKT2 138-457 Fin GDE LCT146 126 (6.273) M1 [Ev354,It2] (Gs,.5,131:1551,2.,L33,R1); Sm (Mn, 2x3 38336. 1 1.83e3 Treated 6M Gu, 1mM EDTA, 2M DTT, 6 o C Column Temp WR AKT2 138-457 LCT157 11 (5.98) M1 [Ev-13181,It2] (Gs,.5,972:1237,2.,L33,R1); Sm (Mn, 2x3 38336. 1 1.1e4 FL Protein FL Protein % +~59 Da (Ni?) 38398. 38414. + Phos % 38414. + Phos 38458. 38478. 3831. 3836. 38436. 38452. 382 383 384 385 mass 382 383 384 385 3 mass

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

PB Erb4999 no tr 6C LCT78 466 (8.547) Sm (Mn, 3x5.); Cm (453:59) 831.54 1 727.79 877.51 727.46 831.2 941.38 979.56 191.16 728.12 % 825.54 115.5 Proteins that don t fly! Standard Conditions TOF MS ES+ 42 651.75 1181.91 1263.35 149.7 LCT69 475 (8.712) Sm (Mn, 3x5.); Cm (456:515) 831.52 1 727.78 941.38 135.47 727.45 831.19831.85 TOF MS ES+ 32 Treated with 6M Guanidine % 651.72 728.12 89.55 115.41 1219.19 1261.21 146.6 1462.81 1523.7 1589.95 1741.23 LCT152 12 (6.163) Sm (Mn, 3x5.); Cm (112:141) 831.66 1 831.31831.96 962.89 938.21 988.9 176.5 1219.4 136.41 146.84 Column Temp = 6 o C TOF MS ES+ 289 % 727.89 727.56 623.99 1523.98 159.23 1741.58 1828.6 1924.8 6 8 1 12 14 16 18 2 22 24 26 28 32 m/z

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

hfas Theoretical MW = 274,629 Da 8 x1 2.5 + TIC Scan FAS_h5.d 1 1 2 1.5 1.5.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 Counts vs. Acquisition Time (min) Measured MW = 274,631 lock mass 5 ug (2pmol) of total protein injected Courtesy of Jon Williams, GSK 34

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

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