Wolf D. Lehmann studied at the Universities of Bonn and Z?rich before gaining his PhD in 1975. He is currently based at the German Cancer Research Center in Heidelberg, Germany. His areas of expertise include analytical biochemistry, liquid chromatography, mass spectrometry, protein analysis, covalent protein modifications, and peptide sequencing by tandem mass spectrometry. He has published 120 peer-reviewed articles, made numerous book contributions and is on the Advisory Board of the FEBS Journal. He is also a member of the Society of German Chemists, German Society for Mass Spectrometry and American Society for Mass Spectrometry.

Protein Phosphorylation Analysis by Electrospray Mass Spectrometry

A Guide to Concepts and Practice

By Wolf D. Lehmann

The Royal Society of Chemistry

Copyright © 2010 Wolf D. Lehmann
All rights reserved.
ISBN: 978-0-85404-185-5

Contents

Acknowledgements, xiv,
Chapter 1 Introduction, 1,
Chapter 2 Analysis of Phosphopeptides by Mass Spectrometry, 18,
Chapter 3 Phosphopeptide Enrichment, 141,
Chapter 4 Dephosphorylation, 191,
Chapter 5 Protein Phosphorylation and Element Mass Spectrometry, 212,
Chapter 6 Structural Phosphorylation Analysis, 234,
Chapter 7 Quantitative Protein Phosphorylation Analysis, 286,
Outlook, 362,
Subject Index, 364,

CHAPTER 1

Introduction

1.1 The Rise of Protein Phosphorylation in the Life Sciences

1.1.1 Reversible Covalent Protein Modification

Enzyme-catalyzed reversible covalent protein modification is a widespread cellular event, which effects that the number of individually distinct human protein species is at least one order of magnitude in excess of the number of gene-encoded proteins. Reversible protein phosphorylation catalysed by kinases and phosphatases is probably the most abundant and most important protein modification principle. In addition, several other protein modifying/ demodifying systems are found (e.g. the pairs acetylase/deacetylase, N-methyltransferase/N-demethylase). Side-by-side to the principle of covalent modification, the functional diversity of the proteome is further expanded by the formation of noncovalent complexes exhibiting a unique functionality, activity or cellular localization. Proteins may form homo- and heteromeric complexes as well as noncovalent complexes with metabolites, drugs or metals. The lifetime of a noncovalent protein complex, its localization and its function may be influenced by covalent modifications. Thus, the interplay of covalent modification and noncovalent interaction forms the basis for the eclectic abilities of the proteome as sensor and regulator element in cells. Figure 1.1 gives a graphic summary of this situation.

Annotation of the human genome resulted in the discovery 518 protein kinase and 214 protein phosphatase genes. This means that about 3% of our genome-encoded proteins are functionally specialized for adding a phosphate group to or removing it from proteins including the kinases themselves. As a result, it is estimated that about 30% of the human proteome is phosphorylated in some way. In addition experimental data allow the estimation that phosphoproteins are on average phosphorylated at three sites. Therefore, protein phosphorylation is probably the most abundant reversible protein modification and it is probably the functionally most diverse and influential one.

1.1.2 The Phosphorus Content of a Cell

Phosphorus is a relatively rare element, which occurs in the biosphere mainly as phosphate. Due to its restricted availability and its multiple biochemical functions, phosphate is often a growth-limiting factor. A eukaryotic cell on average contains 0.5 pmol of phosphorus, mainly in the form of organic phosphate. Its distribution over the most important compound classes of a cell is schematically displayed in Figure 1.2.

Lipids contain about one third of the phosphorus content of a cell followed by roughly equal portions (of around 20%) located in metabolites, RNA, and proteins. ATP/ADP and DNA each contain about 5% of total cellular phosphorus, whereas inorganic phosphate (Pi) represents only about 2%.

1.1.3 Nutritional Proteins Recognized as Phosphoproteins

The phosphate content of proteins first became evident in the characterization of acidic nutritional proteins such as casein and vitellin, which at that time were named conjugated proteins. The next progress occurred when phosphoserine was identified as component of vitellin, a protein isolated from egg yolk. The nutritional protein casein is highly phosphorylated, which confers calcium-binding properties to it. In this way both calcium and phosphate are provided for bone formation. The main constituent of bone is calcium hydroxyapatite, which is a mixed calcium hydroxide phosphate. The recognition of nutritional phosphoproteins as sources for phosphate and calcium describes the first known function of phosphorylated proteins.

1.1.4 Protein Phosphorylation Controls Metabolic Enzymes

The efforts of Carl and Gerti Cori in the 1940’s, and the subsequent studies of Earl Sutherland, Edwin Krebs and Edmond Fischer paved the way for the revolutionary insight, that phosphate attachment was not only an add-on to nutritional proteins, but may control the function of a key enzyme of energy metabolism: glycogen phosphorylase. This enzyme cuts α-D-glucose from glycogen liberating it as α-D-glucose-1-phosphate by concomitant attachment of inorganic phosphate. The Coris unravelled that glycogen phosphorylase is regulated in its activity by hormones and that it exists in two interconvertible forms, an active and an inactive form. Studying the action of hormones on glycogen metabolism, Sutherland discovered the role of cAMP as the first ‘second messenger’ in transmembrane signal transduction. Roughly in parallel to these investigations, Krebs and Fischer discovered the principle of reversible phosphorylation as the metabolic control mechanism underlying the activation/deactivation of glycogen phosphorylase. They found that phosphorylation at Ser15 activates glycogen phosphorylase, whereas dephosphorylation led to inactivation. An important analytical step in this discovery was tryptic digestion of glycogen phosphorylase labeled with 32P and isolation of the radiolabeled peptides. The main radiolabeled peptide found was sequenced as KQI-pS-VR. In these years it was also discovered that kinase-catalyzed phosphorylation requires ATP and Mg2+ ions and that kinase-catalyzed reactions can be performed in vitro. Numerous historical and scientific details of this development are summarized in the corresponding Nobel lectures [http:// nobelprize.org], for instance in those of Krebs and Fischer.

1.1.5 Protein Kinase Cascades as Principle of Cellular Signal Transduction

In the structural characterization of kinases two milestones were achieved using cAMP-dependent protein kinase A as model enzyme. It was the first kinase, to be completely sequenced and for which the complete 3D structure was unravelled. As an extension of phosphorylation at serine and threonine, phosphorylation at tyrosine was discovered and viral oncogene products were identified as tyrosine kinases. In the following, a class of receptors with intracellular tyrosine kinase activity was found, laying the ground for unravelling a widespread mechanism for transmembrane signal transduction. In prokaryotes a signal transduction pathway was discovered based on phosphorylation at histidine (phosphoric acid amide) and at aspartate (phosphoric acid ester) (for reviews see e.g.) which represent two chemically more labile forms of phosphate addition compared to the phosphoric acid esters at serine, threonine and tyrosine.

The effect of phosphorylation is amplified, in case a kinase is modified so that its activity is switched on or off. For instance, in a multistep kinase cascades, one kinase activates the next kinase by phosphorylation, so that an exponential amplification of the start event is achieved. Since kinase cascades are the central principle of many signalling cascades, phosphorylation is one of the keys for transformation of extracellular signals into intracellular responses, as mediated for example by the class of receptor tyrosine kinases. Quenching of these signals is achieved by the action of tyrosine phosphatases, which are a large subgroup of phosphatases.

1.1.6 Structural Consequences of Protein Phosphorylation

As evident from the discovery of protein phosphorylation in the context of glycogen phosphorylase regulation, addition or removal of a single phosphate group may switch the activity of a metabolic enzyme on or off. Later, this concept was extended to kinases and phosphatases, and in general to phosphorylation cascades as discovered, for instance, in signal transduction. In addition, the linkage between phosphorylation and noncovalent interaction was recognized. Mechanisms for this linkage are, for instance, that phosphorylation disrupts existing structural motifs or creates new structures. The latter effect can be proven by the observation that phosphosites are occurring frequently in natively disordered protein regions. Upon phosphorylation, an unstructured region may form a stable structure which enables specific noncovalent interactions with specific target proteins. Figure 1.3 schematically summarizes the functions of protein phosphorylation in metabolic regulation, signal amplification and structural reorganization.

In addition to the reversible covalent modification of serine, threonine and tyrosine, chemically more labile modifications in the form of phosphate amides at His or Lys or of phosphate anhydrides with Asp have also been identified, mostly as intermediates in phosphate transfer processes. Finally, the regulation of nucleotide phosphate binding proteins, occurring e.g. in Ras proteins or membrane-associated G-proteins can be regarded as a kind of ‘noncovalent’ protein phosphorylation. In these systems, the guanine nucleotide exchange factors introducing GTP exhibit a kinase-related function, whereas the GTPase activity corresponds to the protein phosphatase function.

1.1.7 Dysregulation of Protein Kinases can Cause Cancer

One of the molecular mechanisms of cancer development is that genomic mutations hijack a signal transduction pathway, which then provokes a dys-regulated overshooting cellular proliferation. The recognition of viral oncogene products as tyrosine kinases or as products interfering directly with intracellular signal transduction opened the way for a broad acceptance of the involvement of kinases and phosphatases in cellular growth control. The most impressive recent example is the recognition that the chromosomal translocation leading to the Philadelphia chromosome is the molecular cause of chronic myelogenous leukemia (CML). The resulting mutated BCR-ABL kinase is constitutively active and its activity is sufficient to drive the overshooting proliferation of white blood cells.

1.1.8 Protein Kinase Inhibitors as Anticancer Drugs

The direct consequence of the involvement of kinases in cancer is the development of kinase inhibitors as anticancer drugs. These inhibitors may act by different mechanisms, such as by interfering competitively with ATP binding or by stabilizing the inactive conformation of a kinase. Currently, a set of strategies is applied for the development of new kinase inhibitors for cancer treatment. One of the most successful achievements in this field is the introduction of imatinib as highly specific inhibitor of the BCR-ABL kinase for the treatment of CML. This success further stimulated the worldwide search for drugs targeted to aberrant signal transduction processes. It is estimated that currently about 1/3 of pharmaceutical research investments is related to the development of kinase inhibitors for targeted therapies of cancer and other diseases.

1.2 The Rise of Mass Spectrometry in the Life Sciences

In the last century numerous technological innovations occurred in originally separated fields, such as mass spectrometry, liquid chromatography, protein sequencing and computer technology. At a certain level of performance and refinement, these technologies developed a synergistic potential which lifted their analytical results to a new level of productivity and significance giving rise to the field of analytical proteomics. The field of phosphoproteomics exemplifies this development, since here mass spectrometric results may directly unravel biological functions. The dynamics of the research on protein phosphorylation is shown in Figure 1.4, displaying the number of publications retrieved from the publicly accessible scientific literature database PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) using the keywords ‘mass spectrometry’ and ‘phosphorylation’. It can be recognized that this field began to flourish already about 5 years after the introduction of ESI (1984) and MALDI (1987/1988).

Some milestones of the development of mass spectrometry into a key technology of protein and protein phosphorylation analysis are summarized below.

1.2.1 The Start-Electron Impact Ionization

About a century ago J. J. Thomson and F. W. Aston developed in Cambridge, UK, the principle of mass spectrometry (MS). They generated beams of electrically-charged particles in evacuated glass tubes. By exposing them to electric or magnetic fields, they recognized that besides negatively charged electron beams, there were also ‘rays of positive electricity’, which as we now know were atomic or molecular ions. By mass spectrometry the isotopic nature of the elements was discovered, which enabled the development and refinement of the atomic model. Numerous accurate masses of elements were determined by mass spectrometry using high resolution double focusing mass analysers, data which were highly useful for the advancement of the field of nuclear physics. Ionization of gaseous molecules by a beam of electrons (electron ionisation, EI) was the standard ionisation method in mass spectrometry up to the 1960’s. EI is very efficient, but generates ions with high internal energy and a radical site (one electron is lost during ionization), so that for organic molecules, ionisation is in general connected with extensive fragmentation. Structural analysis of mammalian steroid hormones was one of the first successful applications of mass spectrometry in the life sciences. Through their common cholesterol-derived steran system of four annealed rings, steroid hormones show a reasonable stability under electron impact and small structural differences can be read from their fragment ions, so that steroid hormone identification can be performed by EI-MS. From the late 1930’s to the 1950’s the influence of hormones on mammalian reproduction was a central theme in the life sciences. In this context the first orally-active steroid hormone was synthesized and later the birth control pill was introduced. In the late 1950’s and early 1960’s the first mass spectral databases were published as books, containing a large number of EI spectra of low-molecular weight natural compounds. As mentioned above, EI spectra are fingerprint-like mass spectra composed mainly of fragment ions. This is the combined result (i) of the transfer of electronic excitation energy during the ionisation by an electron beam, (ii) of the labile character of the radical molecular ions initially formed, and (iii) of the thermal energy the analytes have acquired during vaporization. Major analytical limitations of EI are that the molecular ion signals of organic compounds are often missing and the necessity of analyte evaporation, so that analytes with poor volatility show EI spectra containing both thermal and ionization-generated fragments. Derivatization sometimes alleviates the situation but numerous organic analytes, such as biopolymers, cannot be tackled at all by EI-MS. These limitations initiated several attempts to develop softer ionisation techniques.

1.2.2 The Intricate Ways towards Soft Ionisation Methods

The first soft ionisation method, chemical ionisation (CI), was developed in an American Oil Company, which at that time made use of EI-MS for characterization of their raw materials and products, both being extremely complex mixtures of hydrocarbons. The aim was to develop MS ionization methods with less fragmentation for a better molecular fingerprinting. The developed CI method solved this task by EI of a reactant gas followed by secondary gas-phase reactions of reactant ions with analyte molecules. CI effects a more soft ionization than EI, but it still requires a transfer of the analyte into the gaseous state prior to ionisation.

The invention of CI marks the start for two vivid decades in the development of soft ionisation methods. Field ionisation (FI) was the next innovation. Here very soft ionisation is achieved through an extremely high electric field of a positively charged wire with activated surface, which detaches electrons from gaseous analytes. However, analytes have to be evaporated before analysis and the molecular ions formed are radicals. Separate evaporation is avoided in the related technique field desorption (FD), where the sample is adsorbed directly on the emitter surface. Heating of the emitter makes the analytes mobile so that they move to the ionizing centres. By FD a very soft ionization is achieved and mostly ‘even electron ions’ of the type [M + H]+ or [M + Cat]+ (Cat = singly charged metal ion) are produced, which exhibit much less fragmentation than ‘odd electron ions’ carrying a radical site. However, in FD the continuous sample supply to the ionizing centres emerged as a major limitation in particular for polar compounds. From then on the development proceeded into two main directions. One was based on discontinuous (sputtering) techniques applied to solid or liquid samples and in the other line; continuously operating ionization techniques were created.

(Continues…)Excerpted from Protein Phosphorylation Analysis by Electrospray Mass Spectrometry by Wolf D. Lehmann. Copyright © 2010 Wolf D. Lehmann. Excerpted by permission of The Royal Society of Chemistry.
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