1 DETERMINING THE SPECIFICITY OF PEPSIN FOR PROTEOLYTIC DIGESTION A thesis presented by Melissa H. Palashoff to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Master of Science in the field of Chemistry Northeastern University Boston, Massachusetts August 2008 2 DETERMINING THE SPECIFICITY OF PEPSIN FOR PROTEOLYTIC DIGESTION by Melissa H. Palashoff ABSTRACT OF THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry and Chemical Biology in the Graduate School of Arts and Sciences of Northeastern University, August 2008 3 ABSTRACT Pepsin is an aspartic acid protease that is commonly found in the stomach of many organisms. Porcine pepsin is the most studied and is fully active at pH 1.9 but inactive above pH ~7. Pepsin is known to have limited specificity and there are only general rules about its cleavage preferences. To further define rules regarding pepsin specificity, a database was constructed consisting of 40 proteins and 1344 peptide cleavages from the literature. Contemporary scientific literature was searched for all publications that involve pepsin digestion and mass spectrometry at pH 2.5-2.7. Peptide data for 40 proteins were extracted and combined to create a map of pepsin cleavage specificity. The frequency of cleavage for each protein was normalized based on how many times that specific combination of residues occurred in the protein sequence. In addition to the literature search, nine proteins along with E.coli whole cell lysate were digested at pH 1.0, 2.5 and 4.0. The proteins were analyzed with online pepsin digestion using an immobilized pepsin column and UPLC/ESI-MSE. The peptides and their fragments were identified with a combination of MSE, software analysis, and manual inspection. The analysis of the data indicated that pepsin maintains limited cleavage preferences. At pH 2.5, pepsin will cleave preferentially after most bulky, hydrophobic amino acids such as leucine and phenylalanine. Additionally, the residues that most often occur immediately following the cleaved peptide bond are tryptophan and tyrosine. It has also been shown that pepsin will rarely cleave at proline and histidine. Analysis performed at pH 1.0 and 4.0 yielded similar results. 4 ACKNOLEDGEMENTS I would like to begin by thanking my advisor Dr. John R. Engen for all of his help in making me a better scientist. Without his guidance and advice this research would not have been possible. I am also grateful to my other committee members, Dr. Mary Jo Ondrechen and Dr. Paul Vouros, for helping to make this thesis the best that it could be. I would like to sincerely thank everyone in Dr. Engen’s lab for their continuous support over the past year. To my labmates, Dr. Thomas Wales, Dr. Roxana Iacob, Christopher Morgan, Sean Marcsisin, Damian Houde and Susan Fang, for providing a wonderful environment for me to work and learn in. Finally, I would like to dedicate this to my family and friends for their constant encouragement during the past five years. I would especially like to thank my parents, William and Patricia, and my brother Joshua for everything they have done to support me, both morally and materially, during my college career. Last, but certainly not least, to Eddie for always being there to support me during the good days and the bad, thank you. 5 TABLE OF CONTENTS ABSTRACT ………………………………………………………………………………………. 3 TABLE OF CONTENTS …………………………………………………………………………. 5 LIST OF FIGURES ……………………………………………………………………………….. 8 LIST OF TABLES ………………………………………………………………………………...10 LIST OF ABBREVIATIONS …………………………………………………………………… 11 CHAPTER ONE: INTRODUCTION AND BACKGROUND TO ASPARTIC ACID PROTEASES AND PEPSIN…………………………………………………………….. 13 1.1 Aspartic Acid Proteases ……………………………………………………………...13 1.2 Catalytic Mechanism of Aspartic Proteases …………………………………………. 15 1.3 Primary Structure of the Pepsin-like Family ………………………………………… 17 1.4 Pepsin …………………………………………………………………………………18 1.4.1 History of pepsin ……………………………………………………………18 1.4.2 Activation of pepsin ………………………………………………………...18 1.4.3 Pepsin crystal structure ……………………………………………………..21 1.4.4 Activity of pepsin ………………………………………………………….. 23 1.4.5 Pepsin and proteomics ……………………………………………………... 23 1.5 Research Objectives …………………………………………………………………. 24 1.6 References …………………………………………………………………………… 25 CHAPTER TWO: LITERATURE SEARCH …………………………………………………… 29 2.1 Introduction ………………………………………………………………………….. 29 2.2 Materials and Methods ………………………………………………………………. 30 2.3 Construction of Cleavage Database …………………………………………………..30 6 2.4 Data Normalization ………………………………………………………………….. 34 2.5 Cleavage Data Map ………………………………………………………………….. 36 2.6 Revised Cleavage Data Map ………………………………………………………… 41 2.7 Summary of Literature Research ……………………………………………………. 44 2.8 References …………………………………………………………………………… 44 CHAPTER 3: EXPERIMENTAL DETERMINATION OF PEPSIN SPECIFICITY AND THE EFFECTS OF pH ………………………………………………………………………... 78 3.1 Introduction ………………………………………………………………………….. 78 3.2 Instrumentation ……………………………………………………………………….78 3.2.1 UPLC and online pepsin digestion ………………………………………… 79 3.2.2 Mass Spectrometry ………………………………………………………… 81 3.3 Materials and Methods ………………………………………………………………. 83 3.3.1 Protein sample analysis ……………………………………………………. 83 3.3.1.1 Protein sample preparation ………………………………………. 83 3.3.1.2 UPLC methods …………………………………………………... 85 3.3.1.3 Mass analysis ……………………………………………………..87 3.3.2 E.coli sample analysis ……………………………………………………... 87 3.3.2.1 E.coli sample preparation ………………………………………... 87 3.3.2.2 UPLC analysis …………………………………………………… 88 3.3.2.3 Mass analysis ……………………………………………………..88 3.4 Data Analysis …………………………………………………………………………88 3.4.1 Software processing ……………………………………………………….. 88 3.4.2 Peptide analysis ……………………………………………………………. 89 7 3.5 Results ……………………………………………………………………………….. 92 3.6 References …………………………………………………………………………...100 CHAPTER 4: PERSPECTIVES AND FUTURE DIRECTIONS ……………………………... 141 4.1 Discussion and Conclusions ………………………………………………………... 141 4.1.1 Literature search ………………………………………………………….. 141 4.1.2 Experimental research ……………………………………………………. 141 4.1.3 Research on pepsin specificity …………………………………………… 142 4.2 Future Directions …………………………………………………………………… 142 8 LIST OF FIGURES Figure 1.1 Aspartic proteases family tree ………………………………………………………... 14 Figure 1.2 The aspartic protease catalytic mechanism …………………………………………... 16 Figure 1.3 Sequence alignment of porcine pepsinogen and porcine pepsin ……………………... 19 Figure 1.4 Crystal structure of human pepsin …………………………………………………… 22 Figure 2.1 Example of a peptic digest map ……………………………………………………… 31 Figure 2.2 Example of cleavage nomenclature …………………………………………………...33 Figure 2.3 Equation used for data normalization and example calculation ……………………... 38 Figure 2.4 Cleavage data map …………………………………………………………………… 40 Figure 2.5 Cleavage data map with probability defined as a percentage ………………………... 42 Figure 3.1 Schematic of online pepsin digestion ………………………………………………… 80 Figure 3.2 Schematic of the operation of MSE …………………………………………………... 82 Figure 3.3 Example MS/MS data of hemoglobin ………………………………………………... 90 Figure 3.4 Example MS/MS data of myoglobin ………………………………………………….91 Figure 3.5 pH 2.5 peptic digest map of Abl ……………………………………………………..102 Figure 3.6 pH 2.5 peptic digest map of Albumin ………………………………………………. 103 Figure 3.7 pH 2.5 peptic digest map of Aldolase ………………………………………………. 104 Figure 3.8 pH 2.5 peptic digest map of Amyloglucosidase ……………………………………...105 Figure 3.9 pH 2.5 peptic digest map of β-Lactalbumin ……………………………………….... 106 Figure 3.10 pH 2.5 peptic digest map of Hemoglobin …………………………………………..107 Figure 3.11 pH 2.5 peptic digest map of Myoglobin ……………………………………………108 Figure 3.12 pH 2.5 peptic digest map of Nef ……………………………………………………109 Figure 3.13 pH 2.5 peptic digest map of Ubiquitin ……………………………………………...110 9 Figure 3.14 Cleavage data map pH 1.0 …………………………………………………………... 93 Figure 3.15 Cleavage data map pH 2.5 …………………………………………………………... 94 Figure 3.16 Cleavage data map pH 4.0 …………………………………………………………... 95 Figure 3.17 Cleavage data map with probability defined as a percentage, pH 1.0 ……………….97 Figure 3.18 Cleavage data map with probability defined as a percentage, pH 2.5 ……………….98 Figure 3.19 Cleavage data map with probability defined as a percentage, pH 4.0 ……………….99 10 LIST OF TABLES Table 2.1 Literature search results ……………………………………………………………….. 32 Table 2.2 Cleavage database ……………………………………………………………………... 51 Table 2.3 Sum of cleavages between two residues ………………………………………………. 35 Table 2.4 Possible cleavages between two residues ……………………………………………... 37 Table 2.5 Normalized cleavage data ……………………………………………………………... 39 Table 2.6 Cleavage data with probability defined as a percentage ………………………………. 43 Table 3.1 Proteins used for digestion ……………………………………………………………. 84 Table 3.2 Auxiliary solvents …………………………………………………………………….. 86 Table 3.3 pH 1.0 peptides ………………………………………………………………………. 111 Table 3.4 pH 4.0 peptides ………………………………………………………………………. 121 Table 3.5 pH 2.5 E.coli peptides ………………………………………………………………... 125 Table 3.6 Normalized pH 1.0 cleavage data ……………………………………………………... 93 Table 3.7 Normalized pH 2.5 cleavage data ……………………………………………………... 94 Table 3.8 Normalized pH 4.0 cleavage data ……………………………………………………... 95 Table 3.9 Normalized pH 1.0 cleavage data defined as a percentage …………………………….97 Table 3.10 Normalized pH 2.5 cleavage data defined as a percentage …………………………...98 Table 3.11 Normalized pH 4.0 cleavage data defined as a percentage …………………………...99 11 LIST OF ABBREVIATIONS ACN acetonitrile