Shankaramma Shivaprasad, Ph.D. Robert J. Duff, Ph.D. Jon S. Kauffmann, Ph.D. Protein Sequencing Using High Resolution LC-MS/MS Lancaster Laboratories Lancaster, PA 17602 Biochemistry Department

Introduction Reduced/alkylated/deglycosylated ovalbumin peptide map MS spectrum of the peptide LTEWTSSNVMEER

RT: 5.10 - 35.14 SM: 7B Ovalbum inreduced # 1186-1230 RT: 17.90-18.30 AV: 23 NL: 5.40E6 Schematic representation of sample preparation for LC-MS/MS analysis 24.04 NL: 1.22E8 F: FTMS + p ESI Full ms [400.00-2000.00] 929.99 TIC F: FTMS + p 100 791.37 ES I Full m s z= 2 [400.00-2000.00] 100 95 MS The development of bioanalytical techniques for rapid identification of proteins is important Ovalbumindeglyc 95 osylated 90 y-ions in biopharmaceutical industry. Tandem mass spectrometry (MS/MS) has become a valuable 90 85 85 Desalting Trypsin digestion Reduced/alkylated sample, 80 tool for characterization of biomolecules owing to its reliability, speed and sensitivity. The 80 LC/MS/MS analysis 75 ease of interfacing the MS with liquid chromatography (LC) make mass spectrometry a 75 70 70 28.28 L–T–E–W–T–S–S–N–V–M–E–E–R 1292.38 powerful technique for applications. This study demonstrates a systematic approach for 1. Denaturation 65 65 26.63 28.72 60 30.59 60 949.82 1390.98 obtaining extensive sequence information over the whole length of a protein in a single 1231.17 2. Reduction/ 55 25.76 55 20.33 762.39 844.42 alkylation 50 50 series of experiments using high-resolution and high mass accuracy tandem mass Reduced/alkylated/ 45 b-ions Desalting Trypsin digestion 45 Relative Abundance Relative Abundance Relative spectrometry. deglycosylated sample, 40 22.77 40 673.37 18.08 LC/MS/MS analysis 791.37 35 Deglycosylation 35 31.63 30 30 1090.51 Ovalbumin 17.27 Ovalbumin, a major protein in avian egg-white which belongs to the serpin super-family, was 25 1129.82 778.37 25 z= 3 20 used for the analysis. Ovalbumin comprises of a single polypeptide chain of 385 amino acid 20 15 847.61 15 12.48 z= 4 1582.72 591.97 10 z= 2 residues which has a single carbohydrate chain linked covalently with Asn292. 14.53 16.21 421.76 10 647.39 32.44 13.48 1153.84 5 523.28 5 8.57 9.40 0 411.71 463.19 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 0 m/z Trypsin digestion Non-reduced sample, 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Tim e (m in) LC/MS/MS analysis Figure 3: Mass spectral profile for the trypsin digest of reduced/alkylated/deglysosylated ovalbumin sample. Figure 6: Mass/charge (m/z) profile for the trypsin digest peptide fragment eluting at 18.34 min and a schematic Objective The retention time and mass/charge values are shown. of the most commonly found fragment ions (inlet) that contain the amino acid sequence information is shown.

To develop a rapid technique for the determination of amino acid sequence information and possible post-translational modifications (PTMs) using a high resolution LC/MS/MS method. The approach involves enzymatic cleavage of the protein to a collection of peptides which are then separated by LC and analyzed directly by MS/MS. Non-reduced ovalbumin peptide map MS spectrum of the peptide ISQAVHAAHAEINEAGR MS/MS spectrum of the peptide LTEWTSSNVMEER Ovalbuminreduced # 1233 RT: 18.347 NL: 8.09E2 Ovalbuminreduced # 749 RT: 12.43 AV: 1 NL:3.66E6 RT: 7.99 - 35.55 SM: 7B F: ITMS + c ESI d Full m s2 [email protected] [205.00-1595.00] 28.72 NL: F: FTMS + p ESI Full ms [400.00-2000.00] 1390.98 7.82E7 591.97 100 z= 3 TIC F: FTMS + p 100 ESI Full m s 100 95 [4 0 0 .0 0 - 95 2000.00] MS 95 Ovalbum innonre 90 90 887.45 duced 90 z= 2 85 85 y-ions 85 LTEWTSSNVMEER 80 80 80

75 75 75 LTEWTSSNVMEER 70 70 70 34.90 1575.81 MS/MS 65 65 65 20.89 I–S–Q–A–V–H–A–A–H–A–E–I–N–E–A–G–R 738.04 32.44 60 60 60 1153.84 23.97 55 55 762.07 REE 55 50 50 +1 +1 yy 3 yy 50 7 45 433.3 864.5 +1 45 yy 9 Relative Abundance Relative Relative Abundance Relative b-ions 1053.5 45 27.66 40 +1 yy 10 Relative Abundance 930.49 40 1239.5 yy +1 40 35 8 35 952.4 30 35 RE 30 LTEW 25.76 33.71 1104.58 25 +1 +1 1176.94 yy 2 bb 5 +1 30 25 +1 bb 9 304.1 bb 631.2 20 4 +1 1019.4 530.1 yy 6 25 21.48 31.90 20 +1 bb 6 778.3 897.91 1157.84 +1 15 718.5 yy 11 16.10 15 +1 20 444.23 bb 10 +1 1368.5 LTE +1 421.76 z= 4 10 bb bb 11 898.44 8 1150.5 bb +1 1279.5 12 10 z= 2 +1 920.0 15 bb 3 1409.0 +1 15.86 5 yy 12 344.0 421.76 30.85 523.28 1183.60 1469.6 5 909.94 1269.83 z= 2 z= 3 10 13.44 z= 2 0 22.86 523.29 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 17.22 20.26 673.37 0 5 9.18 11.04 444.79 844.42 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z 515.33 479.29 m/z 0 Figure 7: MS/MS Spectrum for the tryptic digest fragment with m/z 791.36. The sequence with residues 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Tim e (m in) Figure 4: Mass/charge (m/z) profile for the trypsin digest peptide fragment eluting at 12.48 min and a schematic identified as b-ions (in blue) and y-ions (black). Residues highlighted in red were not found. of the most commonly found fragment ions (inlet) that contain the amino acid sequence information is shown. Representative sequence for b , b , y and y fragment ions is shown. Figure 2: Mass spectral profile for the trypsin digest of non-reduced ovalbumin sample. 3 4 2 3

MS/MS spectrum of the peptide ISQAVHAAHAEINEAGR Results and Conclusions Protein Sequencing Ovalbuminreduced # 739 RT: 12.342 NL: 5.08E2 F: ITMS + c ESI d Full ms2 887.46@ cid35.00 [230.00-1785.00] Confirmation of the sequence was accomplished by LC-MS/MS analysis combined with data processing ISQAVHAAHAEINEAGR 100 and sequence similarity database searching tools. Extensive sequence coverage was achieved with the ISQAVHAAHAEINEAGR 95 +1 yy 10 1068.5 reduced/ alkylated/deglycosylated sample (86%) compared with the non-reduced sample (97%). The 90 +1 +1 yy yy 11 12 GSIGAASMEF CFDVFKELKV HHANENIFYC PIAIMSALAM VYLGAKDSTR TQINKVVRFD 85 1139.7 1276.7 method demonstrates the advantage of fast chromatography combined with high resolution, high 80

75 mass accuracy mass spectrometry to confirm sequence identity of proteins. The method is simple, KLPGFGDSIE AQCGTSVNVH SSLRDILNQI TKPNDVYSFS LASRLYAEER YPILPEYLQC 70 +1 TM bb 8 rapid and useful approach for the characterization of complex proteins. The method can be extended Figure 1: Thermo Scientific LTQ XL -Orbitrap instrument 65 778.5 VKELYRGGLE PINFQTAADQ ARELINSWVE SQTNGIIRNV LQPSSVDSQT AMVLVNAIVF 60 for characterization of unknown proteins sharing identical peptides with related database sequences. (LC/MS/MS) used for the analysis 55 50 KGLWEKTFKD EDTQAMPFRV TEQESKPVQM MYQIGLFRVA SMASEKMKIL ELPFASGTMS 45 Relative Abundance Relative RGAENI 40 +1 bb 6 +1 yy 6 35 636.3 MLVLLPDEVS GLEQLESIIN FEKLTEWTSS NVMEERKIKV YLPRMKMEEK YNLTSVLMAM 659.3 +1 yy 9 30 997.4 +1 bb 10 25 986.6

RGAEN References: +1 +1 GITDVFSSSA NLSGISSAES LKISQAVHAA HAEINEAGRE VVGSAEAGVD AASVSEEFRA 20 yy bb 9 +1 +1 7 +1 bb

Experimental ISQAV yy 14 5 916.3 bb +1 789.2 12 yy +1 yy 3 546.3 1229.6 13 1472.8 15 +1 1375.8 303.1 ISQA bb 5 499.4 Srebalus-Barnes C.A. and Lim A. Applications of mass spectrometry for the structural characterization 10 +1 TM +1 +1 +1 bb bb 4 bb bb 15 16 DHPFLFCIKH IATNAVLFFG RCVSP 13 +1 1601.0 The analysis was carried out on high resolution LTQ XL Orbitrap (Thermo Electron Corp., 5 400.4 1343.7 yy 14 1543.5 1446.9 of recombinant protein pharmaceuticals. Mass Spectrometry reviews 26 (2007) 370-388. TM 0 Waltham, MA) hybrid mass spectrometer in conjunction with an Accela high-speed 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z chromatography system. Mass spectral data was acquired using high mass accuracy full Figure 5: MS/MS Spectrum for the tryptic digest fragment with m/z 887.46. The sequence with residues Huntington J.A. and Stein P.E. Structure and properties of ovalbumin. J. Chromatogr. Ovalbumin sequence: Sequence Coverage for trypsin digested ovalbumin (residues in red) : 97.1% coverage scan and normal accuracy data dependent product ion spectra. All the data was analyzed identified as b-ions (in blue) and y-ions (black). Residues highlighted in red were not found. Representative B 756 (2001) 189-198. sequence for b , b , y and y fragment ions is shown. using Bioworks TM software to produce amino acid sequence information. 4 5 4 5