ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2013

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1059

Enrichment and Separation ofPhosphorylated Peptides onTitanium Dioxide Surfaces

Applied and Fundamental Studies

ANNA I. K. ERIKSSON

ISSN 1651-6214ISBN 978-91-554-8717-1urn:nbn:se:uu:diva-204723

Dissertation presented at Uppsala University to be publicly examined in The Svedbergssalen,BMC, Husargatan 3, Uppsala, Friday, September 27, 2013 at 10:00 for the degree of Doctorof Philosophy. The examination will be conducted in English.

AbstractEriksson, A. I. K. 2013. Enrichment and Separation of Phosphorylated Peptides on TitaniumDioxide Surfaces: Applied and Fundamental Studies. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 1059. 52 pp. Uppsala. ISBN 978-91-554-8717-1.

Protein phosphorylation is a very common posttranslational modification (PTM), which latelyhas been found to hold the keyrole in the development of many severe diseases, includingcancer. Thereby, phosphoprotein analysis tools, generally based on specific enrichment of thephosphoryl group, have been a hot topic during the last decade.

In this thesis, two new TiO2-based on-target enrichment methods are developed and presentedtogether with enlightening fundamental results.

Evaluation of the developed methods was performed by the analysis of: custom peptides, β-casein, drinking milk, and the viral protein pIIIa. The results show that: i) by optimizing theenrichment protocol (first method), new phosphorylated peptides can be found and ii) by theaddition of a separation step after the enrichment (second method), more multi-phosphorylatedpeptides, which usually are hard to find, could be detected. The fundamental part, on the otherhand, shows that the phosphopeptide adsorption is caused by electrostatic interactions, in generalfollows the Langmuir model, and the affinity increases with the phosphorylation degree. Here,however, the complexity of the system was also discovered, as the adsorption mechanism wasfound to be affected by the amino acid sequence of the phosphopeptide.

Keywords: Posttranslational modification, Phosphorylation, Mass spectrometry, MALDI,Adsorption, QCM-D

Anna I. K. Eriksson, Uppsala University, Department of Chemistry - BMC, Box 576,SE-751 23 Uppsala, Sweden.

© Anna I. K. Eriksson 2013

ISSN 1651-6214ISBN 978-91-554-8717-1urn:nbn:se:uu:diva-204723 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-204723)

Till min familj!

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Eriksson, A., Bergquist, J., Edwards, K., Hagfeldt, A.,

Malmstrom, D., Hernandez, V. A. (2010) Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Using Mesoporous Titanium Dioxide. Analytical Chemistry, 82(11): 4577-4583.

II Eriksson, A., Bergquist, J., Edwards, K., Hagfeldt, A., Malmstrom, D., Hernandez, V. A. (2011) Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Se-paration of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry. Analytical Chemistry, 83(3): 761-766.

III Eriksson, A. I. K., Edwards, K., Hagfeldt, A., Hernandez, V. A. (2013) Physicochemical Characterization of Phosphopeptide/ Titanium Dioxide Interactions Employing the Quartz Crystal Microbalance Technique. Journal of Physical Chemistry B, 117(7): 2019-2025.

IV Eriksson, A. I. K., Bartsch, M., Bergquist, J., Edwards, K., Bergström Lind, S., Hernandez, V. A. On-target Titanium Dio-xide-based Enrichment for Characterization of Phosphoryla-tions in the Adenovirus pIIIa Protein. Submitted.

Reprints were made with permission from the respective publishers.

Contents

1 Introduction ......................................................................................... 11 1.1 Protein phosphorylation .................................................................. 11

1.1.1 Posttranslational modifications .............................................. 11 1.1.2 The phosphorylation process ................................................. 12

1.2 Phosphorylation analysis ................................................................. 13 1.3 Phosphopeptide enrichment techniques .......................................... 15 1.4 Aim of this thesis ............................................................................ 16

2 TiO2 – phosphoryl group interaction theory ........................................ 17 2.1 Structural properties ........................................................................ 17

2.1.1 Titanium dioxide .................................................................... 17 2.1.2 The phosphoryl group ............................................................ 18

2.2 The binding mechanism .................................................................. 18 2.3 Adsorption isotherms ...................................................................... 19

2.3.1 Adsorption models ................................................................. 19 2.3.2 Isotherm data evaluation ........................................................ 21

3 Techniques ........................................................................................... 22 3.1 Mass spectrometry .......................................................................... 22

3.1.1 MALDI .................................................................................. 23 3.1.2 Modified MALDI targets ....................................................... 23

3.2 QCM-D ........................................................................................... 24

4 Applied studies .................................................................................... 25 4.1 Optimized on-target enrichment protocol ....................................... 25

4.1.1 Spot target enrichment protocol ............................................. 28 4.2 On-target enrichment and separation .............................................. 30

4.2.1 Caseins ................................................................................... 31 4.2.2 Viral protein ........................................................................... 33 4.2.3 Stripe target enrichment and separation protocol .................. 34

5 Fundamental studies ............................................................................ 35 5.1 Degree of phosphorylation .............................................................. 36 5.2 Amino acid sequence ...................................................................... 37

5.2.1 Tryptic peptides ..................................................................... 38 5.3 Origin of interaction ........................................................................ 40

6 Conclusion and future work ................................................................. 41

Populärvetenskaplig sammanfattning ........................................................... 43

Acknowledgement ........................................................................................ 47

References ..................................................................................................... 48

Appendix – amino acid structures ................................................................. 52

Abbreviations

Biological short names and other abbreviations

ADP adenosine diphosphate

ATP adenosine triphosphate

BSA bovine serum albumin

EGFR epidermal growth factor receptor

IMAC immobilized metal affinity chromatography

IR insulin receptor

JAK janus kinase

K kemptide

MOAC metal oxide affinity chromatography

mRNA messenger ribonucleic acid

MS mass spectrometry

MS/MS tandem mass spectrometry

PTM posttranslational modification

QCM-D quartz crystal microbalance with dissipation monitoring

RNA ribonucleic acid

TLC thin layer chromatography

Chemical compounds

ACN acetonitrile

DHB 2,-5-dihydrobenzoic acid

EDTA ethylene-diamine-tetraacetic acid

IDA imidodiacetic acid

ITO indium tin oxide

NTA nitriloacetic acid

P phosphoryl group

SA salicylic acid

TFA trifluoroacetic acid

TiO2 titanium dioxide

Physical variables

A area

β viscoelastic parameter

C bulk concentration

Γ adsorbed concentration

Γmax surface saturation concentration

D dissipation

f frequency

h thickness

ka adsorption rate constant

kd desorption rate constant

KA equilibrium association constant

m mass

n overtone

θ fractional surface coverage

ρ density

x mass sensitivity constant

11

1 Introduction

1.1 Protein phosphorylation Phosphorylation, the addition of a phosphoryl group (HPO3) to the side chain of an amino acid, is a posttranslational modification (PTM) of proteins that has been in the spotlight lately. Except for being unusually common, about 33% of all proteins in the cell are phosphorylated at some point during their lifecycle1, and as this is interesting from a biological point of view, it is also medically relevant. Several studies have shown that many untreatable dis-eases, like cancer,2 diabetes,3 and neurodegenerative disorders,4 can be asso-ciated with abnormal phosphorylation in the affected cells. A recent study has also presented that phosphorylation might play a keyrole in the viral infection mechanism.5 Thus, a good analysis tool, one specially developed for phosphorylated proteins, is crucial in order to both understand the origin of and find working treatments for several of the common severe chronic diseases in today’s society.

1.1.1 Posttranslational modifications Posttranslational modifications are chemical changes in the protein structure that take place after the messenger RNA (mRNA) code is translated to the amino acid sequence. They play a keyrole in the complex system that builds life, being the source of cell proteome diversity (Figure 1).

Figure 1. From genome to proteome. Illustration of how the approximately 20,000 coding human genes after transcription increase the information by at least five times, which in turn translates to more than 1,000,000 proteins, due to PTMs.

>20 000codinggenes

>100 000transcripts

>1 000 000proteins

12

PTMs can occur at any time during a protein’s lifecycle and are generally responsible for the protein function, regulating activity, spatial structure, and intracellular location. Currently, several hundred different PTMs have been identified. These can be divided into two main categories: covalent addition of functional groups to the amino acid side chain (phosphoryl, methyl, gly-cosyl, etc.) and proteolytic cleavage of the peptide bond.6-8

1.1.2 The phosphorylation process Chemically, phosphorylation is a reversible reaction with the general func-tion of turning protein activity on or off, which is illustrated in Figure 2. In detail, the forward reaction (green part) occurs between ATP and a free elec-tron pair on one amino acid side chain in the protein and is catalyzed by an enzyme group called kinases. The backward reaction (red part) is, on the other hand, catalyzed by a different enzyme group, phosphatases.3

Figure 2. Schematic illustration of the reversible phosphorylation reaction.

Since many of the twenty amino acids available (see appendix) have polar or charged side chains, there are several that theoretically can be phosphory-lated. In reality, there are nine in total, although only the three most common and stable ones are generally considered in the phosphoprotein analysis.6 These three amino acids, serine (Ser/S), threonine (Thr/T) and tyrosine (Tyr/Y) (three letter/one letter amino acid code), all have an alcoholic side chain that becomes the acceptor in the reaction. Figure 3 shows the chemical structure of a normal and a phosphorylated serine, respectively.

PProtein

P

ATP

ADP

Kinase

ProteinP

Phosphatase

H2O

13

Figure 3. The chemical structure9 of a) the normal serine and b) the phospho-rylated serine with the phosphoryl group marked with a ring.

1.2 Phosphorylation analysis The analysis of PTMs on proteins, including phosphorylation, is known to be challenging due to the fact that they are not regulated by any genes. Instead, the phosphorylation is catalyzed by enzymes, whose activities in turn are regulated by other environmental factors, which means that the result de-pends on the actual situation, and neither when nor where the process will occur is known. Thus, where the protein becomes phosphorylated depends on the current stimuli and thereby varies from time to time, i.e., the protein can form several different phosphorylated versions. In addition to this prob-lem, the cell does not activate more proteins than necessary, which means that only a small fraction of the existing proteins of a certain kind becomes modified, making the amount of protein to work with very low.1,6 This im-plies that the phosphorylation analysis requires both isolation of the few phosphorylated proteins and the possibility to characterize the exact position of the phosphorylation. The first part is generally solved by some sort of enrichment step, which utilizes the specific binding of the phosphoryl group to a material (see chapter 1.3). The second part, on the other hand, needs advanced characterization of the phosphoprotein structure, which is best performed with mass spectrometry (MS) of the corresponding peptides.1 Here, the phosphorylated peptides are found by looking for mass peaks equivalent to the addition of a phosphoryl group (HPO3 = 80 Da). Identifica-tion of the exact position does, however, commonly require additional analy-sis in tandem mode MS (MS/MS). The MS technique is described further in chapter 3.1.10,11

14

Figure 4. Schematic illustration of the two different enrichment techniques. The phosphorylations are marked with P.

The general phosphorylation analysis procedure starts with an enrichment step followed by MS and eventually MS/MS. However, the requirement of peptides in the MS step implies that a digestion step must be added some-where before it. As shown in Figure 4, this can be put in either before or after the enrichment, which means that the enrichment target can vary (pro-tein or peptide) depending on the way that is chosen. Both ways have their limits, but digestion before the enrichment, i.e., peptides as the target, is more common in phosphoprotein characterization analysis and therefore is the focus in the next chapter.12,13

Another feature that is important to mention is that due to the digestion, the phosphorylations will be spread out unevenly on the peptides. Most peptides will get zero or one phosphorylation, although some might get two, or three, or even more. In phosphorylation analysis, the phosphopeptides are com-

digestion enrichment

enrichment

digestion

Protein

15

monly divided into mono-phosphorylated (one phosphorylation) and multi-phosphorylated (two or more phosphorylations).

1.3 Phosphopeptide enrichment techniques The phosphopeptide enrichment techniques can be divided into three main categories: affinity-based, immunoprecipitation, and chemical modifications, where the first is the most widely used and thereby is the one covered in this chapter. An affinity-based enrichment technique is, as the name suggests, based on the phosphoryl-group affinity for a certain material. Even here sev-eral different methods have been developed. This summary will include the three largest.13

The most well-known affinity-based method is immobilized metal affinity chromatography (IMAC). In IMAC, positively charged metal ions (Fe3+, Al3+, Ga3+, Ti4+, Zr4+) are chelated to the stationary phase in a column via acidic ligands (generally nitriloacetic acid (NTA) or imidodiacetic acid (IDA)). The enrichment procedure then utilizes the acidity of the phosphoryl group, i.e., the negatively charged phosphoryl group replaces one of the li-gands on the metal. There are, however, some limitations with this method. Firstly, other acidic compounds, like the acidic amino acids (aspartic acid and glutamic acid) or nucleic acids, can also bind to the metal, causing non-specific enrichment. Secondly, weaker binding mono-phosphorylated pep-tides are commonly lost in the elution. Thirdly, chelating agents as EDTA, with the power to remove several of the ligands, can cut off the metal totally from the column. 12-15

Figure 5. Illustration9 of a) the IMAC enrichment and b) the MOAC enrich-ment material.

The material in the column can also be changed to a metal oxide (TiO2, ZrO2, Fe3O4, Al(OH)3, SnO2, Ga2O3), as in the case of the metal oxide affini-ty chromatography (MOAC) method. MOAC works similarly to IMAC,

16

although here the column is packed with the pure metal oxide, to which the phosphopeptide can bind directly. Figure 5 illustrates the difference between the two methods. Due to the direct binding to the metal oxide, MOAC is less sensitive to chelating agents than IMAC is. MOAC has also shown a higher specificity, although some of the problems with non-specific binding remain. In contrast to IMAC, MOAC is biased towards mono-phosphorylated pep-tides, due to a higher affinity for the phosphoryl group that makes the elution of multi-phosphorylated harder.12-14

Lately, the materials involved in the IMAC and MOAC methods have been used to modify matrix-assisted laser desorption ionization (MALDI) MS targets, in so-called on-target methods. The on-target methods can, unlike the column-based methods, handle small sample volumes and avoid sample loss caused by non-specific binding to columns or tubing. However, since the same materials are generally used, they inherit the limits of the corres-ponding material mentioned above.16-28

1.4 Aim of this thesis The aim of this thesis is to develop an improved titanium dioxide (TiO2)-based on-target enrichment method, compared to the ones currently availa-ble, which can enrich and detect phosphopeptides from biological raw ma-terial. In the first paper (paper I), recently published protocols18-20 are opti-mized, which together with the development of a new target lead to a clear improvement of the enrichment results.

The goal with the studies behind the thesis is, however, to detect as compre-hensive a phosphoproteome as possible with only one analysis. This requires elimination of the current bias problems and in turn a deeper understanding of the enrichment process. Thus, in paper II, a new stripe-shaped target is developed, which can separate the enriched peptides. The separation im-proves the signal of the multi-phosphorylated peptides that have been so hard to find before. In order to be able to develop this method further, fun-damental studies of the phosphoryl group-TiO2 interactions involved in the enrichment process are necessary. In paper III, the quartz crystal microbal-ance with dissipation monitoring (QCM-D) technique is used to analyze the adsorption mechanism through the development of adsorption isotherms for specific phosphopeptides. In this study, the effects of several interesting peptide parameters are investigated together with the origin of the interac-tion.

Lastly, in paper IV, a relevant biological sample in the form of a viral pro-tein is used on the stripe target as a final evaluation of the new method.

17

2 TiO2 – phosphoryl group interaction theory

2.1 Structural properties

2.1.1 Titanium dioxide Titanium dioxide crystals exist in three different structural phases at atmos-pheric pressure: rutile, anatase and brookite. Out of these three, rutile and anatase are the most common phases used in TiO2-based applications. In both these phases, the unit cell is tetragonal and each titanium atom binds to six oxygens (Figure 6).29

Figure 6. The unit cells of rutile and anatase.30

One of the main advantages of TiO2 for the phosphopeptide enrichment ap-plication is that it is thermodynamically stable at a wide pH range. However, that does not imply that the surface is unaffected, since the surface net charge will be dependent upon the pH. The average isoelectric point of tita-nium dioxide lies around pH 6, although it varies a bit depending on phase and particle size.31,32 Above this point, the surface net charge is negative, while below it is positive.

©2011 by The Electrochemical Society

18

2.1.2 The phosphoryl group The term phosphoryl group can be related to many different phosphate-containing structures depending upon the context. In the case of phospho-peptides, it is always a phosphate ester. The chemical structure of the phos-phate ester is shown in Figure 7, which also illustrates that it is a diprotic molecule.

Figure 7. The chemical structure9 of a phosphate ester and the deprotonated versions with the pKa value33 for each reaction.

2.2 The binding mechanism The phosphopeptide enrichment procedure utilizes the structural knowledge presented above, i.e., that the charge of both the phosphoryl group and the TiO2 surface can be regulated with pH. Thus, by setting a pH value of the solution where the two parts have opposite charges (2.15 < pH < 6), an elec-trostatic attractive force should be induced between them, leading to the binding. Studies with small organic phosphates34,35 and phosphonates36-39 have shown that the “phosphoryl groups” like to form a bidentate bond with the TiO2 surface, which includes two of the oxygen atoms in the group (Fig-ure 8).

Figure 8. Illustration9 of the bidentate bond between the phosphoryl group and TiO2.

19

The pH range above must, however, be narrowed a bit since the acidic amino acids (aspartic and glutamic acid) can bind as well if they are charged. The pKa values of the side chains on aspartic and glutamic acid are 3.90 and 4.2733, respectively, which implies that the pH range must be cut to 2.15 < pH < 3.90 in order to avoid unspecific binding.

2.3 Adsorption isotherms Thermodynamically, the enrichment, or adsorption, process is an equilibrium between free and adsorbed compound,

(1)

from which the equilibrium association constant can be stated as

(2)

where ka and kd are the adsorption and desorption rate constants, respective-ly. 40

In adsorption experiments, this equilibrium is commonly illustrated with an adsorption isotherm of the experimental data, which is a plot of the adsorbed concentration (Γ), or fractional surface coverage ( θ = Γ/Γmax), versus the free concentration (C) at a constant temperature. The shape of the isotherm plot can then be translated to a mathematical model that describes the physical adsorption mechanism for that experiment. However, in order to cover all possible adsorption behaviors, a huge number of different models have been developed. These are commonly divided into four categories, depending upon the properties of the surface (homogeneous or heterogeneous) and the probability of adsorbate-adsorbate interactions (significant or not). The next chapters will describe some of the most common models as well as the data evaluation process necessary for finding the right model.40

2.3.1 Adsorption models The most well-known and simple adsorption model is the hyperbolic-shaped Langmuir model. This model presumes ideal adsorption behavior, i.e., that the surface is homogeneous and no significant adsorbate-adsorbate interac-tions occur, and is presented in Equation 3 40

20

1 (3)

where Γmax is the surface saturation concentration and KA is the equilibrium association constant.

For heterogeneous surfaces that contain more than one type of adsorption site, the Langmuir model can be expanded to an n-Langmuir model by sum-ming the original equation. In this model, which is shown in Equation 4, each type of site has its own saturation capacity constant and equilibrium constant.40

, ,1 , (4)

This expansion can, however, only be done if all sites fulfill the Langmuir requirements and behave independently.

In the case of significant adsorbate-adsorbate interactions, the models need to take into account that more than one adsorbate layer can be formed. This implies that inflection points are formed at the saturation point of each layer, which leads to an s-shaped plot that requires more complex equations to fit. On homogeneous surfaces, the Moreau model is the simplest 40

1 2 (5)

where I is the adsorbate-adsorbate interaction parameter. This model can, in a way similar to the Langmuir model, be expanded to heterogeneous surfac-es by summation. Another model commonly used on homogeneous surfaces is the liquid-solid extended BET,40,41

,1 , 1 , , (6)

which differs between the equilibrium association constant for the adsorption on the solid (S) and on the layer of adsorbate (L).

21

2.3.2 Isotherm data evaluation The purpose with the evaluation is to find the model that fits the isotherm data best, i.e., the “true” model. However, in order to even be able to do that, the number of possible models must first be reduced to a manageable amount. For most cases it is enough to find the category of the isotherm to reach this level, but in some more evaluation is necessary. This subchapter will counter the most common tool used to determine the isotherm category: the Scatchard plot. 40 The Scatchard plot (Γ/C versus Γ) originates from the Scatchard linear re-gression of the Langmuir model

(7)

which implies that only ideal adsorption isotherms that follow the Langmuir model will get completely linear Scatchard plots. For isotherms with more than one adsorption site, the plot becomes concavely curved instead, while adsorbate-adsorbate interactions are shown as maximum or minimum in the plot. The advantage with the Scatchard plot is that due to lots of studies, not only the category but several models can be identified from the shape of the plot. The disadvantage, on the other hand, is that the plot can easily be mi-sinterpreted if there are not enough data points, especially in the low concen-tration range where scatter is common.42

22

3 Techniques

3.1 Mass spectrometry Mass spectrometry (MS) is an analytical technique that through separation of the ions corresponding to the sample provides a spectrum over the sample mass/charge ratios. The mass spectrometer is build up of three parts: an ioni-zation source, one (or several) mass analyzer(s), and a detector. In the analy-sis, the sample is first ionized and then transformed into the gas phase before being transported to the mass analyzer that separates the ions by mass on the way to the detector. Some mass spectrometers have several mass analyzers and thereby the possibility for additional analysis of the fragments of a spe-cific ion (tandem MS or MS/MS). From the pattern of the obtained frag-ments, the chemical composition of the ion can be determined. In peptide analysis, this is a common method for establishing the amino acid sequence. It is also useful for finding the positions of posttranslational modifications, like phosphorylations, which gives an extra loss of 98 Da (phosphoric acid) upon fragmentation.43

Figure 9. Illustration of the MALDI ionization technique.

++

+

++

+

+

target

= sample= matrix

massanalyzer

+ -

23

3.1.1 MALDI Matrix assisted laser desorption ionization (MALDI) is an ionization tech-nique that is commonly used in proteomics. The technique, illustrated in Figure 9, is based on a conductive target, onto which the sample is placed and covered with a matrix solution that forms crystals when it dries. In the analysis, the solid matrix/sample surface is fired with a laser that ionizes the sample, which leads to sample desorption and desolvation (exact mechanism not known). The obtained gas-phase sample ions are then transferred further into the mass analyzer.43

3.1.2 Modified MALDI targets The modified targets developed in the studies behind this thesis are based on thin mesoporous TiO2 films (~2-4 µM thick), sintered onto conductive glass slides (25 x 75 mm) coated with indium tin oxide (ITO). Figure 10 shows the design of the two different types of targets, with the spot target to the left and the stripe target to the right.

Figure 10. Sketches of the two target types: a) the spot target and b) the stripe target, with the TiO2 films shown in gray.

1 mm1-2 mm

25 mm

75 m

m

a) b)

24

3.2 QCM-D Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) is a technique based on the inverse piezoelectric effect in quartz. The technique can monitor the adsorption of small masses (down to a couple of nanograms) as well as the dissipative properties (rigidity) of the adsorbed film and has been used to study the adsorption processes in several different biological systems.44-50

For rigid films with small dissipation changes (ΔD), the relationship between the adsorbed mass (Δm) and the change in frequency (Δf) is given by the Sauerbrey equation51

∆ ∆ (8)

where x is the mass sensitivity constant (17.7 ng cm-2 Hz-1 at f0 = 5 MHz), A is the active area of the sensor, and n is the overtone number. This relation-ship is, however, only valid if the following conditions are fulfilled: the ad-sorbed mass is smaller than the mass of the quartz crystal, the adsorbed film is rigid, and the adsorbed molecules are evenly distributed on the sensor. Thus, in the case of non-rigid, viscoelastic, films, another model must be used. The following model, developed by Voinova et al.52, can be applied for the quantitative determination of the adsorbed mass in viscoelastic films

∆ Im 2

(9)

∆ Re

where β is a parameter dependent on f0 and the ratio of the viscosity/elastic modulus of the film, while h0 and ρ0 are the thickness and density of the quartz crystal.49,50,52 The solutions to this model are generally obtained with specialized software, like QTools.53

25

4 Applied studies

This chapter summarizes the results obtained from the applied studies, start-ing with the first enrichment trials on the spot target (paper I), then continu-ing with the combined enrichment and separation experiments on the stripe target with caseins (paper II), and a relevant viral sample (paper IV).

4.1 Optimized on-target enrichment protocol For the purposes of this paper, a novel enrichment method was developed by the optimization of the target used in earlier published protocols.18-20 The target, consisting of spots of pure anatase (chapter 3.1.1), was then evaluated by the analysis of three different types of samples: i) model peptide mix-tures, ii) pure tryptic β-casein digest, and iii) tryptic β-casein digest mixed with other peptides in excess. β-casein was chosen due to the well-characterized phosphoproteome and relatively high phosphorylation rate.

In the first part, mixtures of a model peptide named kemptide (RLLASLG) and its phosphorylated version (RLLA-pS-LG) in molar ratios of 10/1, 100/1, and 1000/1 were analyzed. By optimizing earlier reported protocols, i.e., utilizing the knowledge that

• the anatase structure have recorded higher phosphoryl-group enrichment performance than the other phases, 20

• washing with substituted carboxylic acids (DHB), prevent non-specific binding of acidic compounds, 54 and

• phosphoric acid in the matrix enhances the phosphopeptide sig-nal,55

down to as little as 3 fmol of the phosphopeptide could be identified. The protocol is presented in detail in chapter 4.1.1.

This protocol was then successfully used in the next two parts with β-casein as the standard protein. The results of the second part are shown in Figure 11, where the mass spectra of tryptic β-casein both with (b) and without (a) enrichment are presented.

26

Figure 11. Mass spectrum of tryptic β-casein on the spot target obtained a) before and b) after enrichment.

As can be seen in the figure, the amount of phosphopeptides detected in-creased drastically through the enrichment. In total, ten different casein phosphopeptides (both α and β) were identified (Table 1), which is more than had been found earlier with similar methods. Three of the peptides could even be found twice through doubly charged peaks.

In the third part, the same β-casein digest as used above was mixed with digested bovine serum albumin (BSA) or kemptide in excess. The results showed that several of the phosphopeptides detected above still could be detected after the addition of BSA digest (up to ten fold excess) or kemptide (130 fold excess). These experiments did, however, also show how the diffi-culty of the analysis increases with the complexity of the sample, which is illustrated in Figure 12.

1000 1500 2000 2500 3000 35000

20

40

60

80

100

Re

lativ

e in

ten

sity

(%

)

m/z

10

a)

1000 1500 2000 2500 3000 35000.0

2.5

5.0

7.5

10.0

100

Rel

ativ

e in

tens

ity (

%)

m/z

1514

13

12

9

7*

8

54

1

23 6

10

11*

b)

27

Figure 12. Mass spectrum obtained after enrichment of a) a 1/1 mixture of BSA/tryptic β-casein (2.5 pmol) and b) a 130/1 mixture of kemptide (K)/ tryptic β-casein (2.5 pmol). BSA phosphorylated peptides are labeled as BSA-#. Kemptide and its oligomers are labeled as K-#.

Due to the high amount of different non-phosphorylated peptides in the di-gestion, out of which several can be acidic and bind non-specifically, the washing in Figure 12a seems to be incomplete even though the excess is much less than in the case of b. The two numbered BSA peaks correspond to two phosphorylated BSA peptides (#1 = VPQV-pS-pT-P-pT-LVEVSR, #2 = KVPQV-pS-pT-P-pT-LVEVSR).

Another interesting feature found in this study was the enrichment of two methionine oxidized peptides (peak 7 and 11 in Figure 11). At the time there were some uncertainties regarding the origin of this feature: actual enrich-ment or creation by the laser in the MALDI. This was, however, recognized

1000 1500 2000 25000

25

50

75

100

Rel

ativ

e in

tens

ity (

%)

m/z

a)

1

6 83 9

10

BSA-1

BSA-2

1000 1500 2000 2500 3000 35000

25

50

75

100

K-3

K-2

Rel

ativ

e in

tens

ity (

%)

m/z

b)

K

K-1

1 6 89

10

13 15

28

later by Marondedze et al., who used TiO2 as a way to actually study the methionine oxidation PTM and successfully enriched methionine oxidized peptides.56

4.1.1 Spot target enrichment protocol This chapter describes the details of the enrichment protocol developed for the spot target. All experiments were performed in room temperature and atmospheric pressure. The explanations for the chemical abbreviations can be found on page 9.

1. The sample was dissolved in either DHB buffer (20 mg/mL DHB in ACN/H2O/TFA 50/49.9/0.1 (v/v)) or 0.01% (v/v) TFA (pH 2.6, aq).

2. About 3 pmol of the sample was loaded on the spot, either at once or in aliquots, depending upon the conditions. If the sample was loaded in aliquots, the spot was washed once by pipetting 0.5 µL DHB buffer on and off 30 times before the next aliquot was add-ed.

3. The loaded solution was left to enrich for 30 minutes in a humid chamber after each addition before being left to dry after the final one had enriched.

4. The spot was washed thoroughly ten times with DHB buffer, each time the same way as in point 2, and was then left to dry.

5. Finally, the following three solutions were added, all on a dry spot: 0.5 µL of 400 mM NH4OH, 0.3 µL of 2% (v/v) TFA (aq), and 0.5 µL DHB matrix (20 mg/mL DHB in ACN/H2O/H3PO4 50/49.9/0.1 (v/v)).

29

Tab

le 1

. Sum

mar

y of

the

resu

lts o

btai

ned

from

the

case

in a

naly

sis

in p

aper

I a

nd p

aper

II.

The

pep

tides

iden

tifie

d ar

e nu

mbe

red

(num

ber

or

lett

er)

acco

rdin

g to

the

corr

espo

ndin

g pa

per.

[M+

H]+

po

siti

on

pept

ide

sequ

ence

β-

case

in

spot

(n

o.)

β-ca

sein

st

ripe

(n

o.; s

ecti

on)

mil

k sp

ot

(no.

)

mil

k st

ripe

(n

o.; s

ecti

on)

1466

.6

α-S

2 (1

53-1

64)

T

VD

ME

-pS

-TE

VF

TK

3

- -

-

1594

.7

α-S

2 (1

53-1

65)

TV

DM

E-p

S-T

EV

FT

KK

5

- -

-

1660

.8

α-S

1 (1

21-1

34)

VP

QL

EIV

PN

-pS-

AE

ER

6

- 1

1; 2

, 7-9

1832

.8

α-S

1 (1

04-1

19)a

YL

GE

YL

IVP

N-p

S-A

EE

R

- -

- 6;

3

1952

.0

α-S

1 (1

19-1

34)

YK

VP

QL

EIV

PN

-pS

-AE

ER

8

- 2

2; 2

-3

1979

.8

α-S

2 (4

0-56

) N

MA

INP

-pS

-KE

NL

CS

TF

CK

9

- -

-

2061

.8

β (3

3-48

) F

Q-p

S-E

EQ

QQ

TE

DE

LQ

DK

10

B

; 5-8

3

3; 2

, 9-1

1

2431

.0

β (3

0-48

) IE

KF

Q-p

S-E

EQ

QQ

TE

DE

LQ

DK

12

D

; 7

- -

2556

.1

β (3

3-52

) F

Q-p

S-E

EQ

QQ

TE

DE

LQ

DK

IHP

F

13

C; 5

-7

- -

2965

.2

β (2

-25)

E

LE

EL

NV

PG

EIV

E-p

S-L

-pS

-pS

-pS

-EE

SIT

R

14

- -

4; 2

3122

.3

β (1

-25)

R

EL

EE

LN

VPG

EIV

E-p

S-L

-pS-

pS-p

S-E

ESI

TR

15

A

; 2

- 5;

2

a Cau

sed

by a

ltern

ativ

e sp

licin

g54

30

4.2 On-target enrichment and separation

These studies are based on the stripe target method developed in paper II (chapter 3.1.2), which combines phosphopeptide enrichment and separation. The developed method consists of three steps: enrichment, wash, and elu-tion, as illustrated in Figure 13. As visualized in the figure, the separation is performed with a thin layer chromatography (TLC)-like technique by elution of 0.1 M NH4H2PO4 (pH 4.6) through the mesoporous TiO2 structure. In this step, the presence of phosphoric acid in the eluent causes desorption of the enriched phosphopeptides and as the liquid moves up through the stripe they become separated according to their relative TiO2 affinity. The protocol is presented in detail in chapter 4.2.3.

Figure 13. Illustration of the separation procedure. First, the phosphopeptides are enriched on one of the stripe ends. Second, the stripe is washed so that only the enriched peptides remain. Third, elution with NH4H2PO4, which causes the separation.

The MALDI MS analysis was then made by dividing the target into 15 sec-tions (0.5 cm/section), see Figure 14, which were analyzed separately. Each section was treated exactly the same way in all studies.

Enrichment Elution

Wash

31

4.2.1 Caseins In paper II, the newly developed stripe method was evaluated for the first time by using the now well-characterized β–casein. The result of the separa-tion shows that the identified peptides (Table 1) were spread out on the stripe according to Figure 14, which illustrates a clear separation between the mul-ti-phosphorylated peptide (A) and the mono-phosphorylated peptides (B-D).

Figure 14. Picture of the stripe target (in actual size) with the positions of the phosphopeptides identified (A-D) after the separation. The sections are marked as well as the loading position (L).

In addition, as a way to raise the complexity of the sample an additional step, casein originating from low-fat drinking milk was also analyzed. Here, the advantage of the separation step became even clearer, since the detected amount of phosphopeptides increased (Table 1). Figure 15 shows the mass spectra obtained without enrichment, after enrichment (spot target), and at the loading point of the stripe target after enrichment and separation.

1 5 10 15

A C B

DL

32

Figure 15. Mass spectra obtained from drinking milk a) without enrichment, b) with enrichment on the spot target, and c) the loading (section 2) on the stripe target.

1000 1500 2000 2500 3000 35000

25

50

75

100

Rel

ativ

e in

tens

ity (

%)

m / z

13

a)

1000 1500 2000 2500 3000 35000

25

50

75

100

Rel

ativ

e in

tens

ity (

%)

m / z

12

3

b)

1000 1500 2000 2500 3000 35000

25

50

75

100

Rel

attiv

e in

tens

ity (

%)

m / z

1

2

3

4* 5*

c)

33

4.2.2 Viral protein In paper IV, a relevant biological sample was used for the first time. The sample, consisting of the pIIIa protein originating from the Adenovirus type 2 particle, was produced and purified in house before analysis on both the spot and the stripe target. In contrast to the caseins that have been used be-fore, this sample has a low phosphorylation degree and exists in limited quantities. Here, some small adjustments of the enrichment protocols were necessary due to the low available amounts of this sample. The adjustments implied sample loading by the addition of several aliquots, which in turn led to the requirement of washing between the additions on the spot in order to not block the small surface (see protocol in chapter 4.1.1). The results ob-tained after both MS and MS/MS analysis are presented in Table 2, which shows that the detection of three new phosphorylation sites could be verified together with the first-ever findings of multi-phosphorylated peptides.

Table 2. Verified and potentially identified phosphopeptides from the pIIIa studies. Possible phosphorylation sites are marked with lowercase letters.

# Mw

(obs)

Mw

(calc)

#

HPO3*

Position Sequence Verified

(Y/N)

1 1477.9 1477.7 1 447-460 RPssLsDLGAAAPR Y¤

2 1511.1 1512.7 1 336-349 DGVtPsVALDMtAR Y+

3 1626.0 1625.0 2 251-262 DtyLGHLLtLyR Y+

4 1677.3 1677.6 1 51-65$ VsEPLDtsHGMLALK Y+

5 2016.3 2016.2 2 461-478 sDAssPFPsLIGsFtstR Y¤

6 2279.2 2279.1 0 284-304 ALGQEDTGSLEATNYLLTNR N

2279.1 1 503-522 NLPPAFPNNGIEsLVDKMsR¤ N

7 2354.5 2353.1 1 484-502 LLGEEEyLNNsLLQPQREK Y¤

* Number of phosphorylations. ¤ Contains phosphorylation sites reported and validated by Lind et al. (underlined5, bold57). Observe that all previously characterized sites had only been detected in separate spectra of monophosphorylated peptides. + Verified as phosphorylated by MS/MS sequencing in this study. $ From the pIV protein.

Despite the good results of several new phosphorylations, the exact position could not be determined for any of the peptides due to poor peptide fragmen-tation or too many possible site combinations.

34

4.2.3 Stripe target enrichment and separation protocol This chapter describes the details of the enrichment and separation protocol developed for the stripe target. All experiments were performed in room temperature. The explanations for the chemical abbreviations can be found on page 9.

1. The sample was dissolved in either DHB buffer (20 mg/mL DHB in ACN/H2O/TFA 50/49.9/0.1 (v/v)) or 0.01% (v/v) TFA (pH 2.6, aq).

2. About 2-6 pmol of the sample was loaded on one of the stripe ends, ~ 1 cm from the edge, either at once or in aliquots, depend-ing upon the conditions.

3. The loaded solution was left to enrich for 30 minutes in a humid chamber after each addition before being left to dry after the final one had enriched.

4. The whole stripe was then immersed into SA buffer (18 mg/mL SA in ACN/H2O/TFA 50/49.9/0.1 (v/v)) and washed for 30 mi-nutes.

5. The stripe was then moved to a bath of 0.15% (v/v) TFA (aq) for another 30 minutes before being taken up and left to dry.

6. The dry stripe was then put vertically, with the loading end point-ing downwards, into an elution chamber filled with ~ 5 mm of elution buffer, 0.1 M NH4H2PO4 (pH 4.6) (see Figure 13).

7. After 15 hours, the stripe was removed from the chamber and left to dry horizontally before being wetted with 2% (v/v) TFA (aq), left to dry again, and then finally being covered with DHB buffer prior to the analysis (phosphate already included in the elution buffer).

35

5 Fundamental studies

This chapter summarizes the results obtained from the fundamental studies in paper III together with some unpublished data.

The studies in this chapter were performed with pure phosphopeptides on titanium-coated QCM-D sensors, oxidized by piranha treatment (3:1 sulfuric acid:hydrogen peroxide). The peptides analyzed were chosen specifically for the investigation of the following parameters:

1. Degree of phosphorylation 2. Amino acid sequence 3. Origin of the interaction

All peptides used in these studies are presented with name, sequence, degree of phosphorylation, average charge, and chapter in Table 3 below.

Table 3. List of the peptides studied in this chapter.

name sequence degree of

phos.

average charge

(pH 2.6)chapter

OnepY ESYVESYVES-pY-V 1 -1 5.1

TwopY ESYVES-pY-VES-pY-V 2 -2 5.1

ThreepY ES-pY-VES-pY-VES-pY-V 3 -3 5.1

EGFR DADE-pY-LIPQQG 1 -1 5.2, 5.3

JAK VLPQDKE-pY-pY-KVKEPGE 2 +1 5.2, 5.3

IR TRDI-pY-ETD-pY-pY-RK 3 0 5.2, 5.3

EGFR-R DADE-pY-LIPQQR 1 0 5.2.1

EGFR-2R RADE-pY-LIPQQR 1 +1 5.2.1

EGFR-S-R DADE-pS-LIPQQR 1 0 5.2.1

EGFR-S-2R RADE-pS-LIPQQR 1 +1 5.2.1

36

5.1 Degree of phosphorylation In this study, the effect of the degree of phosphorylation (number of phos-phorylations per peptide) on the adsorption mechanism was investigated by the analysis of three custom-designed peptides with the same amino acid sequence, but different amounts of phosphorylations (Table 3). Figure 16 shows the raw QCM-D data obtained for one of the peptides.

Figure 16. Example of a QCM-D measurement, with the frequency in black, dissipation in gray, and bulk concentration shown for each step.

The relative scales of the frequency and dissipation in Figure 16 imply that the film is rigid and that the Sauerbrey relationship can be applied, which also was the case for the following studies. Adsorption isotherms (example shown in Figure 18) were then created by plotting the surface coverage frac-tion (θ, calculated by dividing the obtained Δf values by the maximum cov-erage frequency, Δfmax) versus the equilibrium bulk concentration (C). Re-markably, the results of the isotherm analysis show that the adsorption me-chanism of all three peptides, no matter the phosphorylation degree, can be explained by the simple Langmuir model. Thus, there is only one adsorption site for the phosphoryl-group on TiO2, and the adsorbate-adsorbate interac-tions are not significant for these peptides. By applying the Langmuir model onto the adsorption data, the equilibrium association and kinetic constants could be calculated; these are shown in Figure 17.

37

Figure 17. Diagrams of the changes in the a) equilibrium association con-stant, b) rate of adsorption, and c) rate of desorption, by degree of phosphory-lation.

An interesting feature that can be seen in Figure 17 is that the association constant increases with a factor of two for each degree of phosphorylation, while the adsorption rate constant remains the same. Thereby, it seems like the differences of the affinity depend upon the desorption rate constant.

5.2 Amino acid sequence Peptides occurring from real samples will, however, not have the same se-quence. Thereby, in the second part of this study, three commercially availa-ble peptides, EGFR (1pY), JAK (2pY) and IR (3pY) (Table 3), are studied in order to find out if the amino acid sequence can affect the adsorption me-chanism. The obtained results showed that the first two peptides (EGFR and JAK) followed the same adsorption mechanism as the three custom ones above, i.e., the Langmuir model, and association constants within the same range according to their degree of phosphorylation were obtained. The third peptide (IR) did, however, show a completely different mechanism. Figure 18 shows the adsorption isotherms (left) and Scatchard plots (right) obtained for the three-times phosphorylated peptides, ThreepY and IR (see Table 3). The inflection point obtained in the IR isotherm, also visualized as the max-point of the sad-mouth shape in the Scatchard plot, indicates that there are significant adsorbate-adsorbate interactions (positive cooperativity).

1 2 30

2

4

6

8

10

12

14

KA (

µM

-1)

Degree of phosphorylation

a)

1 2 30

1

2

3

4

5

6

c)b)

k a/1

0-3 (

s-1 µ

M-1)

Degree of phosphorylation1 2 3

0

2

4

6

8

10

12

14

16

k d/1

0-4 (

s-1)

38

Figure 18. Adsorption isotherms (a) and Scatchard plots (b) obtained for the two three-times phosphorylated peptides studied.

Evaluation of the data showed that the best fit was obtained with a two-layer extended BET model, developed by Gritti et al.58

, 2 , ,1 , , , (10)

where KA,S is the equilibrium association constant for the surface coverage, i.e., the mono-layer formation, while KA,L is the equilibrium association con-stant for the building of the second layer. The results of the fit did, however, surprisingly show that KA,L became much larger than KA,S, which did not even reach the value obtained for the mono-phosphorylated EGFR.

Regarding the origin of this new behavior, the most plausible explanation pointed towards the charge distribution of the IR peptide. Unlike the other peptides studied so far, IR is tryptic-like (i.e., ends with R or K) and has a relatively large amount of positively charged amino acids positioned at the ends, which might interact electrostatically with the phosphoryl-group on another peptide. Further investigations of the adsorption of tryptic peptides are covered in the following subchapter.

5.2.1 Tryptic peptides In this study, the adsorption mechanisms of four custom-designed tryptic versions of the EGFR peptide (Table 3) were analyzed in order to find out the requirements necessary for positive cooperativity. The parameters checked were:

• phosphorylated amino acid (S or Y), • amount of charged ends (1 or 2), • average charge (0 or +1).

0.01 0.1 1

0.0

0.2

0.4

0.6

0.8

1.0

IRThreepY

θ

C (µM)

a)

0.0 0.2 0.4 0.6 0.8 1.0

0

2

4

6

8

10

IR ThreepY

θ /C

(µ

M-1)

θ

b)

39

Figure 19 shows the three different types of Scatchard plots that were ob-tained from the adsorption measurements performed in this study. Each plot consists of data points from several independent measurements.

Figure 19. Types of Scatchard plots obtained from the tryptic EGFR versions. The legend shows which type each peptide answer to as well as the peptide sequence with the charged amino acids in bold.

Both plot a (EGFR-R) and b (EGFR-S-R) show cooperativity trends, though they look very different. Plot a got a tail- shaped end, which indicates that the adsorption mechanisms at low and high concentration are different. This is not seen in b, but here were the low concentration points were omitted because of large uncertainties in the experimental data. It is important to notice that plot a and b corresponds to the two peptides with only one argi-nine, while plot c, which has a linear shape (dotted line), answers to the two peptides with arginine in both ends (EGFR-2R and EGFR-S-2R). Thus, it seems like the cooperativity is not dependent upon the amino acid phospho-rylated (both Y and S show cooperativity) and can only be found for tryptic peptides with an average charge of zero, i.e., one charged end. These results thereby suggest that positive cooperativity might be common among tryptic mono-phosphorylated peptides, considering no missed cleavages in the pep-tide sequence.

EGFR-R (DADE-pY-LIPQQR)

EGFR-S-R (DADE-pS-LIPQQR)

EGFR-2R (RADE-pY-LIPQQR)

EGFR-S-2R (RADE-pS-LIPQQR)

40

5.3 Origin of interaction In this part, the origin of the phosphopeptide-TiO2 interaction was studied. If the theory presented for the phosphoryl group (Chapter 2.2) can also be ap-plied to phosphopeptides, the interaction should mainly be electrostatic and thereby possible to screen by the addition of ions (salt). Thus, EGFR, JAK, and IR were studied with the addition of 25 mM NaClO4. The results, pre-sented in Figure 20, show a clear decrease in the equilibrium association constant for all peptides, even in the layer-layer constant of IR. This con-firms the theory of a mainly electrostatic origin of the phosphopeptide-TiO2 interaction and is another indication that the layer-layer interaction of IR is electrostatically driven.

Figure 20. The effect of salt on the equilibrium association constants for EGFR, JAK, and IR. In the case of IR, both the surface-layer (S) and layer-layer constants are shown. Observe the break in the y-axis.

0 mM 50 mM0

1

2

3

4

5

40

80

KA (

µM

-1)

[NaClO4]

EGFR JAK IR (S) IR (L)

41

6 Conclusion and future work

The studies performed in this thesis show how the on-target TiO2-based me-thods can be improved and how important it is to know the theory behind the interaction utilized. It is, however, necessary to mention that there is still a lot to do, both on the applied and the fundamental parts, before the theory is completely understood or the methods developed can detect a comprehen-sive phosphoproteome from complex, real biological samples. This chapter will summarize the conclusions that can be drawn so far and give some input on what is left to do in future studies.

The results from the applied studies show that the TiO2 bias can be reduced by the addition of a separation step in the method (stripe target), which in-creases the signals from the multi-phosphorylated peptides. The later method also seems to be able to detect more phosphopeptides in total from complex samples. This might be due to the limited size of the spot or the differences in the washing procedures (pipette/bath). Further, the studies also show that the achievement of a good result requires not only the optimization of the basic protocol (chemicals, pH, time, etc.), but also adjustments depending upon the sample studied (amount available, solvent, etc.). Regarding future work, the stripe method could be improved further by optimizing the:

• washing (find the thin line between too much and too little) • size of the stripe (can be made both thinner and longer) • elution (the time is currently long)

In addition, experiments on pure peptides with and without cooperativity would be good in order to fully understand the consequences for the applied part.

The results from the fundamental studies did, on the other hand, really show how complex this system is. Some general properties were found, i.e., in-crease in affinity by degree of phosphorylation and Langmuir adsorption, but due to the findings of cooperativity in net zero charged peptides, the only certain conclusion is that the origin of the interaction is electrostatic. The parameters studied in this thesis are, however, far from all that can affect the enrichment results. For example, it would be interesting to look further into the following factors:

• amino acid phosphorylated (S/T/Y) • position of the phosphorylation (end/middle)

42

• chemicals in the peptide solution (substituted carboxylic acid, phosphoric acid)

• surface microstructure (the target is mesoporous)

43

Populärvetenskaplig sammanfattning

Introduktion Proteiner, en av livets viktigaste beståndsdelar, är mycket komplexa moleky-ler som styr många avgörande funktioner i våra celler. Till exempel är de flesta enzym, hormoner och antikroppar proteiner, liksom hemoglobin som transporterar syre i vårt blod. Proteiner består i grunden av långa kedjor av aminosyror som har länkats ihop på ett visst sätt, förutbestämt av våra gener. Kedjorna veckas sedan ihop till avancerade tredimensionella strukturer som utgör ett fullständigt protein. Proteinet är, dock, inte färdigutvecklat än. De flesta proteiner genomgår en eller flera modifikationer till under sin livscy-kel. Dessa modifieringar, så kallade posttranslationella modifieringar (PTMs), är avgörande för proteinets funktion, då de bland annat reglerar aktiviteten, 3D-strukturen och placeringen inuti cellen. Man har idag hittat flera hundra olika modifieringar, vilka i de flesta fall innebär att vissa delar av aminosyrakedjan klipps bort eller att andra kemiska molekyler adderas.

Figur 1. Fosforgrupp9 och cancercell (med tillstånd från dream designs/ FreeDigitalPhotos.net).

En av de vanligaste modifieringarna är fosforylering; adderingen av en fos-forgrupp (HPO3) till sidokedjan på någon av de tre aminosyrorna: Serine (S), Threonine (T) eller Tyrosine (Y) (se figur 3 i avhandlingen). Just denna pro-cess har uppmärksammats rejält de senaste åren eftersom forskare har funnit att uppkomsten av flera svåra, ej botbara, sjukdomar som cancer, diabetes och demens, kan bero på onormal fosforylering hos viktiga proteiner i de sjuka cellerna. Därmed har också behovet av att kunna analysera det fosfory-lerade proteinet, fosfoproteinet, på ett bra sätt ökat markant.

!

44

Analys av fosfoprotein Analysen av fosfoprotein är, dock, långt ifrån enkel eftersom:

• Posttranslationella modifikationer styrs inte av några gener utan av ”stimuli”, signaler från omgivningen. Var och när ett protein fosforyleras beror på det aktuella stimulit, alltså finns det olika fosforylerade versioner av ett visst protein som uppstår i olika si-tuationer.

• Fosforylering är en reversibel process, dvs. den kan tas bort via en annan reaktion (se figur 2 i avhandlingen).

• Cellen använder inte fler proteiner än som behövs, vilket innebär att endast en väldigt liten del av till exempel protein ”A” blir fos-forylerat.

Därmed krävs det att analyseringsmetoden både kan utskilja de få fosforyle-rade proteinerna från resten samt identifiera den exakta positionen. De meto-der som används idag analyserar oftast proteinets peptider som bildas genom att klippa ner proteinet med ett enzym (digestion, Figur 2).

Figur 2. Illustration över hur ett protein blir till peptider via digestion.

Den genomsnittliga analysmetoden börjar med en berikning av fosfopepti-derna, vanligtvis genom specifik inbindning av fosforgruppen till en yta av metallkomplex eller metalloxid (till exempel titandioxid, TiO2). Därefter analyseras de i en masspektrometer som separerar dem efter deras massa. Resultatet blir ett spektrum som innehåller en pik för varje observerad pep-tidmassa. Med hjälp av databaser och kunskapen om vart det använda enzy-met klipper kan fosforyleringarna sedan detekteras. Även om de flesta meto-der fungerar så är de långt ifrån perfekta och vanligtvis krävs flera försök för att detektera alla fosforyleringspositioner på ett protein. Ett annat problem är att de flesta metoder har svårt för att detektera peptider ur båda de grupper som bildas vid klyvningen: peptider med en fosforgrupp, mono-fosforylerade, och peptider med två eller flera fosforgrupper, multi-fosforylerade.

digestion

protein peptides

45

Nya analysmetoder Målet med den tillämpade delen av avhandlingen var att utveckla en TiO2 baserad on-target metod, speciellt framtagen för masspektrometri med MALDI (en joniseringsmetod), som kan identifiera peptider ur båda grup-perna samt fler fosforyleringspositioner från endast en analys. I artikel I användes tidigare publicerade kunskaper för att optimera en redan befintlig metod. Resultatet visade att optimeringen hade effekt, då fler fosfopeptider kunde detekteras i ett redan välkaraktäriserat prov, β-casein. I artikel II ut-vecklades en helt ny metod, som genom ett separationssteg efter berikning-en, lyckades separera mono- och multi-fosforylerade peptider från β-casein och mjölk, och därmed lösa problemet nämnt tidigare. Metoden evaluerades ytterligare i artikel IV, då ett relevant biologiskt prov i form av ett viruspro-tein analyserades. Detta mer komplexa prov (lägre fosforyleringsgrad och begränsad mängd att tillgå) krävde vissa justeringar i metoden, men resulta-ten visade att tre ny fosforyleringar hade detekterats samt de första multi-fosforylerade peptiderna någonsin.

Teoretiska studier För att kunna förstå de existerande problemen med dagens analysmetoder samt kunna förbättra de presenterade ovan ytterligare, gjordes teoretiska experiment i artikel III, där själva inbindningen av fosforgruppen till TiO2 studerades. Teoretiskt sett borde bindningen ske på grund av positiva elek-trostatiska krafter mellan den positiva TiO2 ytan och den negativa fosfor-gruppen (se Figur 8 i avhandlingen). Resultaten visade att detta var sant samt att den generella fosfopeptiden följer den enklaste adsorptionsmekanismen. De visade också hur attraktionen ökade ju fler fosforyleringar en peptid hade, vilket kan förklara att analysmetoderna märker skillnad mellan mono- och multi-fosforylerade. Dessutom påvisades hur aminosyrasekvensen kan påverka adsorptionsmekanismen, då en studerad peptid med positiva amino-syror i ändarna visade sig ha elektrostatiska interaktionskrafter även mellan sig själv (positiv kooperativitet). Vidare studier kring när detta uppstår visa-de att det troligen krävs att peptiden har en positiv laddning i ena änden samt en netto laddning på noll. Detta är intressant eftersom ett av de vanligaste enzym som används vid proteinklyvning klipper aminosyrakedjan efter två av de positiva aminosyrorna och därmed efterlämnar en positiv ände.

46

Slutsats

Resultaten av studierna presenterade i den här avhandlingen visar att: • Optimering av metoden är viktigt samt att den troligtvis behöver

anpassas efter provet. • Det går att detektera både mono- och multi-fosforylerade peptider

med hjälp av ett separationssteg efter berikningen. • Ytterligare kunskap kring teorin bakom metoden som används är

viktig för vidare optimering av metoderna.

Dessutom krävs fler studier kring när positiv kooperativitet uppstår samt vad det får för effekt för analysresultatet.

47

Acknowledgement

First of all, I would like to thank my head supervisor, Anders Hagfeldt, both for taking me on as a PhD student and for accepting this project that lies so far from dye-sensitized solar cells. It has been a fantastic time that I have enjoyed a lot.

I would also like to thank Katarina Edwards for taking me into her group and giving me the possibility of staying in the biological part of physical chemi-stry at BMC. It has been useful to have you on the other side of the wall. You are always available, and I have had a lot of useful discussions with you.

Especially large thanks goes to my third supervisor, Victor, who has helped me the most and has given me support in this sometimes lonely project.

I also want to thank all of the nice people I have had around me, both at BMC and Ångström. Malin, Karin, Emma, Jonny, Amelie, and Sara in Kata-rina’s group for all the nice time in the lab and chats in the lunch room; Jo-nas and David, for giving me support on the analytical part and helping me run the MALDI; Torgny and Jörgen, for introducing me to the world of ad-sorption isotherms; and all the people in Anders’s group, especially Eva, Hanna, Libby, Erik and Leif, for always being so kind to me the few times I worked in the Ångström lab.

Till sist, ett stort tack till min familj som alltid har trott på mig och stöttat mig hela tiden!

48

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52

Appendix – amino acid structures

The chemical structures of the 20 amino acids9, divided into three groups de-pending on the polarity of the side chain. Each amino acid is marked with their full name as well as the three-letter/one letter code in parenthesis.

Lysine (Lys/K)

Aspartic acid (Asp/D)

Arginine (Arg/R)

Glutamic acid (Glu/E)Histidine (His/H)

Charged side chain

Phenylalanine (Phe/F)Alanine (Ala/A) Tryptophan(Trp/W) Valine (Val/V)

Proline (Pro/P)Methionine (Met/M) Leucine (Leu/L)Isoleucine (Ile/I)

Nonpolar side chain

Glutamine (Gln/Q) Tyrosine (Tyr/Y)

Serine (Ser/S) Cysteine (Cys/C)Threonine (Thr/T)

Glycine (Gly/G)

Asparagine (Asn/N)

Polar side chain

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1059

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