shotgun approach in proteomics
Storms, H.F.
Citation
Storms, H. F. (2007, March 29). Capillary isoelectric focusing – mass spectrometry for a shotgun approach in proteomics. Retrieved from https://hdl.handle.net/1887/11461
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Capillary isoelectric focusing – mass
spectrometry for a shotgun approach in
proteomics
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van de Rector Magnificus, prof.mr. P.F. van der Heijden, volgens besluit van het College voor Promoties
te verdedigen op donderdag 29 maart 2007 klokke 16:15 uur
door
Henricus Franciscus Storms
geboren te Leiden
in 1977
promotor: Prof.dr. J. van der Greef co-promotoren: Dr. U. R. Tjaden
Dr. R. van der Heijden
referent: Prof.dr. H. Irth (Vrije Universiteit Amsterdam)
The printing of this thesis was financially supported by the J.E. Jurriaanse stichting.
ISBN: 978907867503 7
Überzeugungen sind gefährlichere Feinde der Wahrheit als Lügen.
(Dogma’s zijn grotere vijanden van de waarheid dan leugens.) Friedrich Nietzsche
Chapter 1 General introduction and scope 7
Chapter 2 Mass spectrometry for biomolecules 15 1. Developments of mass spectrometry for biomolecules 16
2. Electrospray ionization 17
3. Matrix assisted laser desorption ionization 22
4. Instrumentation 26
5. Peptide sequencing and computer software 28
Chapter 3 Capillary electrophoresis and capillary isoelectric focusing 33 1. Capillary electrophoresis and proteomics 34 2. Development of capillary electrophoresis 34
3. Principles of capillary electrophoresis 35
4. Capillary isoelectric focusing 37
5. ESI–MS interfaces 40
6. Coupling of CIEF to ESI-MS 42
Chapter 4 Capillary isoelectric focusing – mass spectrometry for a 47 shotgun approach in proteomics
Chapter 5 Optimization of protein identification from digests as 63 analyzed by capillary isoelectric focusing - mass spectrometry
Chapter 6 Considerations for proteolytic labeling: optimization of 18O 85 incorporation and prohibition of back-exchange
Chapter 7 Relative quantitation of protein abundance by differential 103 analysis of isotopically labelled peptides using CIEF-MS/MS
Chapter 8 Conclusions and perspectives 113
Samenvatting 117
Curriculum Vitae 123
Chapter 1
General introduction and scope
1.1 Genomics, proteomics and system biology
The living cell is in complexity still far beyond our comprehension. Despite decades of intensive research, we are able to understand only small parts of its working.
However, the general outlines are clear.
At the basis of all cellular processes lies the genetic material. In the early 1950s Watson and Crick unraveled the double helix structure of DNA, revealing the basics of its functioning. Now, well into the genomics era, the sequencing of DNA has provided a fundamental starting point for the understanding of the processes of life and disease. Since the Human Genome Project has been finished1, the task at hand is to determine the function of each gene (“functional genomics”).
Analyzing the genome and detecting mutations and polymorphisms should uncover starting points for drug development.
What is equally important is the understanding of the read-out of DNA: the proteins. Compared to genomics research, science is faced with an even bigger challenge, since the protein content of a cell is by far more diverse. More importantly, it is constantly changing in response to environmental stimuli.
Proteomics is the systematic analysis of proteins and their expression patterns in biological systems. It involves the localization, isolation, identification and functional characterization of the proteins in an organism at a particular point in time. Genome sequence databases greatly facilitated this research area, since these are used for the identification of proteins. This made the boom in the field of protein analysis possible.
However, understanding the genome and proteome is only part of the story.
Science has begun to realize more and more that cellular processes are ruled by a complex interplay between genes, proteins and metabolites. To understand processes underlying diseases, the complete picture has to be considered2. Though for decades drug developers focused on specific ‘targets’ (e.g. genes or proteins), a shift is being made towards the analysis of cells as integrated systems: systems biology. A gene or a malfunctioning protein is often not the single underlying cause of a disease. More often, disease should be considered as an imbalance expressed in multiple biological factors (e.g. read-out of genes, functionality of proteins and concentration of metabolites).
This holistic way of thinking calls for new strategies for identifying drug targets and finding appropriate drugs. Broad profiling on several levels (genome,
proteome and metabolome), biostatistics and bioinformatics are needed to obtain information about the dynamics of the system studied. In this approach,
General introduction and scope genomics, proteomics and metabolomics are not an end by themselves, but tools used in a larger scheme.
1.2 Approaches for proteome studies
Proteomics plays a key role in the systems biology approach. Expression patterns of proteins are essential to obtain an understanding of the systems functioning.
This thesis focuses on the development of new tools for proteomics studies.
The number of expressed proteins present in a single cell is vast: it may vary from a few thousand to 20.0003. Separation of the proteins is essential for their
analysis. Two-dimensional gel electrophoresis (2D-GE) is a very powerful tool for the separation of large numbers of proteins. In one direction, proteins are separated according to their isoelectric point (pI) using isoelectric focusing (IEF), and in the orthogonal direction the proteins are separated according to their molecular mass (Mr) using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). This way, up to 10.000 proteins can be separated in one single gel.
Apart from the development of 2D-GE, the blooming of proteomics wouldn’t have been possible without the rapid developments in the field of mass spectrometry (MS). The development of soft ionization techniques, such as matrix assisted laser desorption ionization (MALDI) and electrospray ionization (EI), eventually
facilitated the analysis of large biomolecules. MS offers high analysis speed, high sensitivity and is able to provide the researcher with an intrinsic property of the analyte molecules: their mass.
The classic approach used for the analysis of complex protein mixtures is relatively straightforward. To separate the proteins, 2D gel electrophoresis is employed.
Then, after detection of proteins of interest, these are digested in the gel by trypsin (which specifically cleaves after arginine and lysine residues) and extracted and subsequently analyzed by MS. The information provided by MS can be
combined with the information from the genome sequence databases. The MS spectrum of a tryptically digested protein can be regarded as a fingerprint, since the masses of the tryptic peptides for every protein in a database can be calculated.
The high separation efficiency of 2D-GE and the analytical capabilities of mass spectrometry have proven to be a very powerful combination in the analysis of complex protein mixtures. Still, there are some serious limitations to this
approach. First of all, the mass range of proteins to be analyzed is limited and the technique is not compatible with hydrophobic proteins as membrane proteins.
Moreover, the dynamic range is limited and the technique suffers from a limited 9
reproducibility. Finally, the 2D-GE is very labor intensive and commonly requires several days.
These limitations have enticed analytical chemists to develop alternative approaches for proteome analysis4,5. In 1997, Opiteck et al.55 described an
approach using liquid chromatography (LC). The proteins were separated by 2D-LC and detected by MS. Though this approach was very promising, it still met some limitations. First of all, the sensitivity –especially for larger proteins- is suboptimal.
Furthermore, the mass of a protein alone does not provide enough information for its identification. Therefore, a flow splitter was used to collect fractions and analyze the proteins off-line after tryptic digestion.
To overcome these limitations, Link et al.4 proposed the so-called shotgun approach (figure 1). Now, the protein extract is digested at the beginning of the work flow, and the resulting peptides are then separated by liquid chromatography (LC) or capillary electrophoresis (CE). MS/MS analysis is used for the identification of the individual peptides and subsequently the corresponding proteins. In the past decade, this approach has become increasingly popular. Because of its high speed and ease to automate, it has been used in various applications.
Figure 1. Shotgun approach for proteome studies. First, proteins are digested by trypsin. Subsequently, the peptides are separated by LC or CE and identified by MS/MS analysis. Finally, database search reveals the identities of the original proteins.
General introduction and scope 1.3 CIEF in proteome studies
Although LC still is the most popular method used, CE has some clear advantages6,7. CE combines high separation efficiency with low sample
consumption. However, the small dimensions of CE also carry a disadvantage.
Because of the limited loadability of the capillary, the sample concentration detection limit is relatively high. Thus, to use the technique to its full potential, some kind of sample concentrating technique has to be applied8.
One possible approach is capillary isoelectric focusing (CIEF). CIEF is a high- resolution separation technique that can be applied to amphoteric compounds, such as proteins or peptides. These are separated according to their pI-values, in a pH gradient formed under the influence of an electric field9. An attractive feature is the simultaneous separation and concentration, in contrast to most other analytical techniques in which dilution is part of the separation process.
On-line coupling of CIEF to MS has proven to be quite a challenge. One of the major limitations is the presence of the carrier ampholytes, used for the
generation of a linear pH gradient. Because of their involatile nature, they cause dramatic suppression of MS signal intensity of the analytes. However, both complex mixtures of proteins and peptides are able to form a pH gradient by themselves, in a process called autofocusing.
This thesis will explore the possibilities of CIEF coupled to MS as an analytical technique for proteome analysis. The autofocusing effect of peptides offers exciting changes. Because of the focusing effect, the peptides are concentrated during analysis. If separation is sufficient, complex samples of protein digests can be analyzed. By means of MS/MS analysis of the peptides, the proteins can be identified rapidly, which would not be possible for undigested proteins.
Though autofocusing of proteins has been performed before10,11, autofocusing of protein digests in combination with MS/MS has never been described. Therefore, we will study the efficacy and the limitations of this approach. This technique would provide several advantages over LC approaches. As already mentioned, the amount of sample needed is considerably smaller. Furthermore, the analyte molecules will be ordered according to their pI value which may provide an extra criterion for the identification of the individual peptides.
1.4 Quantitative proteomics
To unravel the principles governing living cells, identification of the proteins at a particular point in time is not sufficient. Differential analysis -the comparison of
11
two or more samples in a quantitative way- is essential for the understanding of the dynamics of the system analyzed; quantitation is thus a prerequisite.
For a long time, quantification by means of MS was not considered to be a viable method. This is mainly due to ion suppression effects occurring in complex samples and variable ionization efficiencies for different peptides. However, isotopic labeling of peptides provides a solution.
In this thesis, quantitative aspects of proteolytic 18O labeling are studied in relation to CIEF-MS analysis of proteins. Although the combination of 18O labeling with LC has already been described in literature, the combination with CE has never been applied. If this combination is successful, the CIEF-MS approach could be
considered as a valuable, complementary technique to the use of LC in the shotgun approach.
1.5 Outline of this thesis
Because of its importance for this research, several aspects of mass spectrometry of biomolecules are discussed in chapter 2. The two “soft” ionization techniques, MALDI and electrospray, are described and the instrumentation is discussed.
In chapter 3, the principles of CE and CIEF, including the coupling to MS, are described. The use of carrier ampholytes in CIEF causes dramatic suppression in signal intensity of the analytes because of their involatility. Several solutions will be discussed.
In chapter 4, the effects of carrier ampholyte concentrations in CIEF-MS experiments for protein digest analysis are studied, including the possibility of autofocusing occurring in the absence of carrier ampholytes.
The use of strongly reduced concentrations of carrier ampholytes should improve separation efficiency without having serious effects on sensitivity. This is further explored in chapter 5. By analyzing a protein extract from Escherichia coli cells, its applicability for biological samples is tested. Furthermore, various aspects of MS/MS data acquisition and analysis are optimized using a mixture of standard proteins. It is studied whether the theoretical pI values of the identified peptides can help in the process of protein identification.
Chapter 6 discusses theoretical aspects of proteolytic 18O labeling and explores several paths to improve the labeling process. Moreover, the compatibility of 18O labeling with CE and CIEF is addressed, which is complicated by the need to deactivate trypsin. The use of proteolytic 18O labeling combined with CIEF-MS in autofocusing mode is evaluated in chapter 7.
General introduction and scope In chapter 8, general conclusions are drawn concerning the possibilities of CIEF- MS for the use of proteome research and suggestions are given for further research.
References
1. Austin CP (2004) Annu. Rev. Med. 55, 1-13
2. Van der Greef J, McBurney RN (2005) Nat. Rev. Drug Discovery 4, 961-967 3. Celis E, Gromov P (1996) Electrophoresis 10, 16-21
4. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JRI (1999) Nat. Biotechnol. 17, 676-682
5. Opiteck GJ, Lewis KC, Jorgensons JW, Anderegg RJ (1997) Anal. Chem. 69, 1518-1524 6. Hutterer KM, Jorgenson JW (1999) Anal. Chem. 71, 1293-1297
7. Neusüss C, Pelzing M, Macht M (2002) Electrophoresis 23, 3149-3159 8. Moini M (2002) Anal. Bioanal. Chem. 373, 466-480
9. Hjerten S, Zhu MD (1985) J. Chromatogr. 346, 265-270 10. Sova O (1985) J. Chromatogr. 320, 15-22
11. Pospíchal J, Glovinová E (2001) J. Chromatogr. A 918, 195-203
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Chapter 2
Mass spectrometry for biomolecules
2.1 Developments of mass spectrometry for biomolecules The analysis of biomolecules has been greatly facilitated by the developments in mass spectrometry (MS). Since mass spectrometers are able to detect several compounds simultaneously and provide their individual molecular masses, they belong to the most powerful detection systems. An equally important feature is the possibility of structural elucidation of the analyte molecules by analysis of fragmentation patterns.
A mass spectrometer consists of three units: the ion source, the analyzer and the detector. In the ion source, gaseous ions are produced of the analytes. By using electromagnetic fields, these ions can be manipulated in flight, which makes it possible to determine their mass-to-charge ratios. This possibility was first recognized by J.J. Thomson1, who constructed a mass spectrometer in the first decade of the 20th century. Using magnetic and electric fields, he could separate ions of different mass-to-charge ratios (m/z). Thus, he was able to prove that neon was composed of two different isotopes (20 Da and 22 Da), providing the final proof of the atomic theory of matter2.
During the late 1950’s the modern applications of mass spectrometry started to develop. The most common method available for the ionization of the molecules was electron impact: ionization by high energy electrons. With this method, only volatile compounds of small molecular weight could be analyzed.
Electron impact is a ‘high-energy’ method; the ionized molecules obtain a surplus energy. Therefore, electron impact automatically results in fragmentation of the analyzed ions. This gave rise to the development of libraries containing mass spectra of reference compounds, to help with the analysis of the resulting mass spectra. All molecules have a specific fingerprint, provided that the energy of the electron beam used is standardized.
The fragmentation also posed a limit to the complexity of the samples to be analyzed. For unambiguous interpretation of the spectra, it was either needed to analyze samples as pure compounds, or build in a separation step.
The obvious first choice for separation was gas chromatography (GC), since one was dealing with volatile compounds. On top of that, the coupling of GC to MS is relatively straightforward. It was first demonstrated by Golhke3 in 1959 and remains to be very popular.
Coupling of liquid chromatography (LC) to MS turned out to be more challenging.
LC is performed at ambient pressure in liquid medium, while MS detection is performed close to vacuum. Furthermore, compounds analyzed by LC are not necessary volatile, as is the case for GC.
Mass specrometry for biomolecules One of the first attempts to tackle these complications was the moving belt technique. Liquid eluting from the LC was deposited on a moving belt, which transported the sample through a series of vacuum locks from atmospheric pressure to the vacuum region of the ionization source4,5. The moving belt technique was far from ideal: it resulted in loss of resolution concerning the separation, and build-up of background signal because of the difficult task to clean the belt during the analysis6. Apart from these problems, this technique was still not compatible with non-volatile compounds or thermo labile compounds.
Two developments in the late 1980s marked the rise of the use of MS for the analysis of larger biomolecules7: matrix assisted laser desorption ionization (MALDI) on the one hand and electrospray ionization (ESI) on the other hand.
These are soft ionization techniques, providing energy for molecules to ionize, but without providing a great surplus energy resulting in break–up of the molecule.
The scientific impact of both MALDI and ESI has been widely recognized. The pioneering work for the development of electrospray as an ionization technique for mass spectrometry was carried out by Dole et al. in the late 19608,9. However, it was John Fenn who laid the fundamentals for the modern ESI-MS technique, demonstrating its essentials and applying the technique for the ionization of biological macromolecules and polymers10,11.
Fenn was awarded with a Noble Price in Chemistry in 2002, sharing it with Koichi Tanaka, who was the first to successfully apply MALDI for small proteins such as chymotrypsinogen (25,717 Da), carboxypeptidase-A (34,472 Da) and cytochrome c (12,384 Da)12,13. It has to be noted though, that independently of Tanaka et al., Hillenkamp et al. 14 developed MALDI-MS in the same year.
2.2 Electrospray ionization
2.2.1 Principles of ESI
The principle of ESI is displayed in figure 1. A high electric potential applied to a capillary causes the liquid to form fine threads, which break up in small droplets.
Basically, the spray that is formed closes the electrical circuit. Thus, a fine spray of highly charged droplets is formed, resulting in the ionization of the analyte
molecules.
17
Figure 1. Principle of ESI.
For large molecules, ESI leads to multiple charges per ion. This favorably results in lower m/z values and lower demands on the mass spectrometer concerning its mass range. On the other hand, because of the distribution of the ions in multiple charge states, the sensitivity is compromised.
ESI is perfectly suitable as an interface for LC or CE, since ionization takes place directly from liquid phase. Conditions are very mild: under suitable circumstances even whole enzyme-substrate and other non-covalent complexes can be
analyzed15-17.
2.2.2 ESI mechanism
Figure 2 displays the principles of ESI in further detail. The electric field results in charge separation at the surface of the liquid. The positive ions are drawn to the counter electrode, and thus the Taylor-cone is formed. Because of the increasingly high charge density at the surface, a critical point is reached at which coulombic repulsion of the excess positive charge is equal to the surface tension of the liquid: this point is known as the Rayleigh limit18.
Figure 2. Schematic representation of a Taylor cone.
Mass specrometry for biomolecules Now droplets, containing an excess charge, are released from the Taylor cone.
While the droplets move to the orifice of the mass spectrometer, the liquid will evaporate, leading to increased surface charge density. Again, the Rayleigh limit will be reached, resulting in fission of the droplets and the production of smaller droplets.
How gas phase ions are formed is still a matter of debate19. Dole et al.20 proposed the coulomb fission mechanism, which assumes that the above described process -the evaporation of the droplets leading to increasingly high charge density, until the droplet divides into smaller droplets- continues until eventually droplets are formed which contain only one charged ion. After evaporation of the remaining liquid, gas phase ions are produced.
An alternative mechanism is known as ion evaporation, which was proposed by Iribarne and Thomson21. This assumes that the high charge density eventually results in coulombic repulsion: the release of gas phase ions directly from the droplet surface. As Iribarne and Thomson argued, when the droplets resulting from the fission process are small enough (R ≈ 10nm) the droplet charge required for ion evaporation is smaller that that required for coulomb fission.
In their review about electrospray ionization, Cech and Enke19 have identified four processes through which ionization can occur: charge separation, adduct
formation, gas-phase reactions and electrochemical oxidation or reduction.
Charge separation is the most common. Because of the electric field applied, the positively charged ions will be drawn to the surface of the Taylor cone, while negatively charged ions will reside inside the fluid. This way, ionic analytes will automatically become part of the excess charge in the Taylor cone. Organic molecules containing functional basic or acidic groups will be protonated or deprotonated because of the excess charge, resulting eventually in positive or negative gas phase ions.
When salts are present, adduct formation can occur in the solution (with e.g.
sodium, lithium, ammonium, chloride). Polar analytes that contain no acidic or basic groups may profit from the adduct formation22,23. However, too high concentrations of salts may lead to the formation of salt clusters, resulting in increased background and diminishing the main analyte peaks19,24.
Gas phase reactions are especially important when the proton affinity of the analyte molecules in solution differs from the proton affinity in gas phase, which are not necessary related. Within a group of analytes, a change in order of basicity may occur after the compounds have entered the gas phase. Gas phase ions may then act as a proton donor for neutrals, when the latter have higher gas- phase basicity25.
19
This means that the height of the signal in the resulting MS-spectrum is not necessarily related to the pKa values of the analytes, but more likely to the gas- phase proton affinity. Amad et al.26 have evaluated this effect by studying the response of observed H2O cluster ions with a varying amount of methanol present.
Though H2O has higher proton affinity in solution than methanol27, in gas phase this order is reversed (184.9 kcal mol-1 for MeOH, 173.0 kcal mol-1 for H2028).
Already at a MeOH/H2O mole fraction of 0.12, H2O cluster ions were not observed, and even at a mole fraction of 0.06, the methanol clusters were predominant.
In the same research, the effect of solvent gas-phase proton affinity on analyte response was studied using 2,2,2,-trifluoroethanol. They found that, when the gas phase proton affinity of the sheath liquid solvent exceeds the gas phase proton affinity of the analytes, analyte signal is completely suppressed even at high concentration of analyte. However, this is commonly not a problem for the analysis of proteins and peptides, which generally possess high gas-phase basicities19.
The last process through which ion formation can occur is oxidation or reduction.
The electrospray is a part of an electrical circuit: voltage is applied between the mass spectrometer and the spray needle. The current through the power supply will be the same as the current arising from the excess charge moving from the spray needle to the MS-entrance. To sustain this current, electrochemical reactions will occur at the electrical contact from the spray tip29: oxidation when measuring in the positive mode, reduction when measuring in the negative mode.
Since ions are formed from neutral species, this can be considered as an ionization method30.However, the electrochemical reactions might also result in unwanted effects, like the consumption of the analyte in a destructive way.
As stated earlier, ESI results in multiple charges31. Above 2000 Da, singly charged ions are hardly observed. Large biomolecules will often carry a variable amount of charges, resulting in so-called envelopes: for a specific analyte, multiple ions are observed in the mass spectrum, only differing in the number of charges they carry, while adjacent peaks differ by only one mass32.
It was Fenn who first realized that this could be used for the improvement of molecular mass determination33. He posed that different charge states could be interpreted as independent measurements of molecular weight. Another attractive feature is that the m/z range of the mass spectrometer required for the
observation of large biomolecules is reduced.
2.2.3 ESI response
The sensitivity for different compounds when applying ESI is difficult to predict. As described above, the in-solution pKa value of an analyte cannot be used. In the
Mass specrometry for biomolecules final stage of the ionization process, proton-transfer takes place in gas phase, which means that gas-phase proton affinity has to be considered.
Apart from the gas phase proton affinity, another important variable is the surface activity of the molecule. When the affinity for liquid surface is higher, the analyte will provide a higher signal. Iribarne et al.34 were the first to observe an increased response for non-polar analytes. They suggested that non-polar compounds would preferably reside at the droplet surface and enter the gas phase more easily.
It has been shown that, during the process of fissioning of the droplets, no
“explosion” occurs, but that a cone-like shape is formed on the droplet-surface, from the tip of which the smaller droplets are formed35 (figure 3). During this process, the parent droplet loses more of its charge than of its mass36, so the offspring droplets are enriched in charge. The compounds on the surface of the parent droplet will more likely be part of the offspring droplets, and as a result will have a better chance to be charged in the ESI process37.
Figure 3. Offspring droplets formed from a parent droplet.
As a result of environmental conditions, the response for certain analytes can be suppressed, which is called ion suppression. Both sample matrix and coeluting compounds can contribute to this effect38.
At sample concentrations of approximately 10 μM, saturation of the ion signal is observed. Several studies have shown that this is not the result of depletion of charge39-41. However, one explanation was given by Iribarne and Thomson in the ion evaporation model. When the droplet surface is crowded with analyte ions, the passage to the gas phase for the completely solvated ions inside will be blocked, resulting in a nonlinear relationship42.
Similarly, at high concentrations, ion suppression of specific ions is observed.
Several studies suggested that the signals of surface-active ions are less suppressed than highly solvated ions. Furthermore, surface-active ions can suppress the signals of highly solvated ions but not vice versa43,44. This suggests that competition takes place for space or charge. Surface-active ions are likely to out-compete highly solvated ions19. Likewise, several comparative studies found that the presence of non-polar side groups gives more intense peaks in a mass spectrum45.
21
However, the main reason for ion suppression is unfavorable composition of the spray liquid droplets. When less volatile or non-volatile compounds are present, the efficiency of droplet formation and evaporation is compromised46,47. Common examples of these non-volatile compounds are salts or ion-pairing compounds.
Also trifluoroacetic acid (TFA), which is commonly used as ion-pair agent in the LC separation of peptides and proteins, is known to result in ion suppression.
Substituting TFA with weaker acids, such as acetic acid and formic acid, commonly results in improved sensitivity48.
The phenomenon of ion suppression makes separation an essential step in the analysis of complex samples, even though a mass spectrometer can detect multiple compounds simultaneously.
2.3 Matrix Assisted Laser Desorption Ionization
2.3.1 MALDI analysis
The forerunner of MALDI is Fast Atom Bombardment (FAB), in being able to successfully ionize thermally labile biomolecules49. Like ESI, FAB and MALDI are soft ionization techniques. However, as opposed to ESI, both FAB and MALDI lead predominantly to singly charged ions. This makes the mass spectra easier to interpret. On the other hand, the requirements for the mass spectrometer are higher: the mass range should be sufficient for the detection of higher masses.
The principle of FAB is based on literally bombarding the analyte molecules with high energy atoms (e.g. 6 keV Xenon atoms). The analyte is dissolved in a liquid matrix, a non-volatile material (commonly glycerol) that serves to replenish the surface with new sample during the ionization process. Although the mass range is greatly extended as compared to Electron Impact, it was still limited to 12000 Da.
This technique was followed by MALDI. As opposed to FAB, for MALDI the energy transfer takes place in an indirect manner. The analytes are imbedded in a
suitable crystalline matrix; when a laser with appropriate wavelength is shot at the matrix, the matrix molecules absorb the energy, heating them up to phase
transition temperature49, resulting in vaporization and ionization of matrix molecules.
Use of lasers for the generation of ions was first applied in the 1960s50. This technique was only feasible for small biomolecules like oligosaccharides51. Because of transfer of excess energy to the analyte ions, molecules larger than 1000 Da could not be observed52. The breakthrough was achieved in 1987 with the introduction of MALDI by Hillenkamp and Karas14, who applied an organic matrix, and Tanaka et al.12,13, who applied an inorganic matrix.
Mass specrometry for biomolecules The matrix molecules transfer energy to the analytes. On top of that, they are able to act as an electron donor or receptor53. Using MALDI, molecules up to 500.000 Da have been analysed54,55, and the sensitivity is greatly enhanced as compared to FAB. MALDI is a very efficient ionization technique and allows for the measurement of compounds with high accuracy and sensitivity56,57.
One of the limitations of MALDI mass spectrometry is the difficulty to couple it on line with LC or CE, because matrix has to be applied and crystallized. However, compared to ESI, it is relatively tolerant for the presence of contaminants (salts, buffers, detergents). Because of their higher diffusion constants, these smaller molecules are presumably partially excluded from the crystals. Still, it is advisable to perform some sample clean-up since contaminants can result in ion suppression and might interfere with the crystallization process58.
Another advantageous feature is the production of singly charged ions, which makes it ideal for the analysis of complex samples like protein digests59. MALDI is commonly coupled to a Time of Flight (TOF) analyzer, because of its extended mass range.
For MALDI-analysis, sample, imbedded in matrix, is commonly applied on the MALDI-target and presented to the mass spectrometer for analysis. The sample preparation is a key factor to obtain optimal results60,61. There are several ways to apply the sample. In essence, the analyte and the matrix should be mixed
properly and they should co-crystallize. This way, the analyte molecules are imbedded in the crystals. The ratio of the matrix to the sample is a determining factor for a successful measurement. Matrix molecules should be in excess ranging from 1000:1 to 5000:162.
The most frequently applied sample preparation method is the dried-droplet method, in which a small volume of sample and matrix solution are applied onto the MALDI-target and allowed to dry61. This method results in inhomogeneous crystals, and the analyte molecules favorable reside in bigger crystals aggregated at the edge of the drop63,64. As a result, MALDI-users have to search for “hot spots” at the surface.
Apart from the dried-droplet method, there are numerous variations, one of which is the “fast evaporation” method. The matrix solution is applied on the target in a highly volatile solvent (e.g. acetone), resulting in a thin film of matrix, on top of which sample is applied65,66. This results in more homogenous crystals and supposedly increased signal reproducibility and sensitivity67.
Some of the matrices that are commonly used are shown in figure 4. Derivatives of benzoic acid and cinnamic acid have proven to be very effective for proteins68. The efficiency of the MALDI matrix might vary for different samples, since each
23
peptide/protein has a unique structure that needs to be incorporated into a specific matrix crystal lattice. Sinapinic acid is commonly most suitable for proteins and glycoproteins, CHCA for peptides, small proteins, lipids and ligosaccharides, while DHB can be used more universally69.
Figure 4. Frequently used MALDI matrices.
Commonly, lasers with wavelengths in the near-UV region are used, like N2 lasers (337nm) or Nd-YAG lasers (355nm). While the matrix molecules will absorb these wavelengths, most analyte compounds will be non-absorbing in the near-UV70. The energy of the laser will be dispersed as vibrational energy over the matrix, and the crystal integrity will diminish. When matrix is heated up to phase transition temperature, this results in vaporization of matrix molecules71. The ionization of the analytes is a result of acid-base reactions, though the exact mechanism is still under debate.
2.3.2 Ionization processes
The fundamental processes of ionization and desorption are still poorly
understood72. The most frequently used explanation for ions resulting from the MALDI process is a multiphoton ionization process (MPI), which leads to a matrix (M) radical cation73:
M
(hv)
M*n (hv)
M+
.
+ e-M
(hv)
M*n (hv)
M+
.
+ e-Mass specrometry for biomolecules For many matrices, radical cations are observed while also electron are emitted, supporting this model74. If matrix is present as radical cation, hydrogen atom transfer occurs readily to form protonated matrix75.
MH+ + (M - H)- M+
.
MH+ + (M - H)- M+.
Because of the high proton affinity of peptides and proteins, proton transfer to form protonated analyte (AH+) is expected to be very efficient. The time-span during which these reactions can occur (during the life time of the plume) seems to sufficiently long for thermodynamic equilibrium to occur, if laser intensity is high enough to form a dense plume76.
An alternative model, proposed by Karas et al. 72 is called excited-state proton transfer (ESPT). This model requires only single photon absorption, and also postulates a mechanism for the ionization of the analyte. After excitation by a photon, the matrix molecule is presumably more acidic. Ground-state matrix molecules or analyte-molecules will subsequently act as proton acceptor76.
M +
hv
M*M* + A (M - H)-+ AH+ M* + M (M - H)-+ MH+
M +
hv
M*M* + A (M - H)-+ AH+ M* + M (M - H)-+ MH+
Both this model and the above mentioned proton transfer assume gas-phase reactions taking place in the plume. These secondary ionization mechanisms are considered to play an essential role in the ionization process. Proton transfer is the most important secondary reaction58. This is the reason why most analyte classes appear in protonated form, in particular proteins and peptides77. Both matrix- matrix as matrix-analyte reaction may occur.
Concerning ion intensities, several factors have to be considered. First of all, homogenous sample preparation is essential to obtain reproducible results. As stated earlier, the commonly used “dried droplet” method results in
inhomogeneous disposition of analyte molecules imbedded in the matrix.
Furthermore, ion suppression can occur. If sufficient analyte is present, matrix ions can be completely suppressed78. This effect does not seem to be limited to specific combinations of analyte and matrix76. Likewise, one analyte is able to suppress another. Therefore, for tryptic digests, only a limited set of peptides is observed for every protein.
25
These effects are likely to be a result of the secondary
(protonation/deprotonation) reactions occurring in the plume. When these reactions are thermodynamically favorable, they can proceed to completion.
Therefore, when peptide-peptide suppression is studied, this appears to be strongly related to the gas-phase basicity79,80.
2.4 Instrumentation
Since Thomson built the first mass spectrometer, the applications of mass spectrometry has steadily grown. Different analyzers have been developed, serving multiple applications.
2.4.1 Time-of-Flight (TOF)
Time-of-Flight mass spectrometry was first successfully applied by Wiley and McLaren81 in the 1950s. Ions from the ion source are accelerated, using an electric field, to a fixed amount of kinetic energy. Subsequently, they travel through the field-free flight tube for mass analysis (figure 5). Smaller ions (or actually ions with a smaller m/z ratio) will have obtained a higher velocity during the acceleration phase and will arrive at the detector before the larger ones82,83.
Figure 5. Schematic representation of a TOF analyser.
TOF analyzers are characterized by a wide mass range, high sensitivity and high ion throughput. However, TOF mass analysis is limited by a generally low mass resolution, attributed to the uncertainties in the location and the initial kinetic energy distribution of the ions in the source at the time they are pulsed out of the source region into the flight tube.
Mass specrometry for biomolecules Reflectrons (ion mirrors) were introduced in 1961 to compensate for variations in energy distribution82. After the acceleration, ions move trough the first field-free region and will arrive at the ion mirror, which consists of a series of grids. Their paths are now reversed, and the ions will move through a second field-free region. If two ions with the same mass but different initial kinetic energy are desorbed from a surface, the ions with the highest kinetic energy will move deeper into the ion mirror before being reversed. Even though its velocity in the field-free regions is higher, this is compensated by the longer path it has to traverse.
Some of the ions may decay in flight as a result from some surplus energy after the ionization. This so-called postsource decay may be used to obtain structural information from molecules83. Generally, this is of limited value as a tool for peptide sequencing because of its low sensitivity.
2.4.2 Quadrupole and quadrupole ion trap
Quadrupoles consist of four parallel rods with a direct current (DC) potential and a superimposed alternating radio-frequency (RF) potential. Ions moving through the quadrupole will possess an oscillating trajectory, which will only be stable when the m/z ratio matches the electric field imposed. The RF field is varied to bring ions of different m/z into focus on the detector and thus build up a mass spectrum.
The quadrupole was developed by Wolfgang Paul84, who would share a Noble price in Physics in 1989. It is the most common mass spectrometers in existence today. This is largely due to its low cost. On top of that, quadrupoles are capable of routinely analyzing up to a m/z of 3000. This makes them attractive as
electrospray analyzers since biomolecules commonly produce a charge distribution below m/z 3000.
Usually, quadrupole analyzers contain three sections: two analyzing quadrupoles at the ends and a central quadrupole that contains the ions during fragmentation for MS/MS experiments; this configuration is known as triple quadrupoles. Apart from performing MS/MS experiments, they can be programmed for a variety of scan modes85.
The ion trap mass analyzer was, just as the quadrupole, developed by Wolfgang Paul. The principles on which both of these analyzers are based are very similar.
An ion trap consists of one ring electrode and two endcaps. The ions are trapped in a three dimensional electric field. Scanning is performed by injecting all ions in the trap, and changing the RF field in order to eject (and detect) ions one by one86.
27
The ion trap is also suitable for performing MS/MS experiments, by isolating a specific m/z ratio and ejecting all others from the trap. The isolated ions are fragmented by collision with gas and the fragments detected to generate a fragmentation spectrum. The primary advantage of ion traps is that multiple collision-induced dissociation experiments can be performed in series (MSN).
One recent development is the introduction of the linear ion trap. The linear (or 2D) ion trap is similar to the conventional 3D ion trap, but the ions are focused along a line rather than to a point. The change in configuration results, among other things, in increased ion injection efficiency and increased capacity for ions.
The linear ion trap not only shows increased sensitivity, but also a higher scanning speed87.
2.4.3 Ion cyclotron
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) is an increasingly popular technique used in the field of proteomics. Concerning its mass resolution, FT-ICR is unequalled, while it simultaneously provides high mass accuracy and good sensitivity88,89.
The basics of FT-ICR MS were developed by Comisarow et al.90 Similar to the ion trap, FT-MS is capable of storing ions within a cell. It consists of three pairs of parallel plates arranged as a cube, used for trapping, excitation or detection. Ions are subjected to a RF electric field and a uniform magnetic field, resulting in spiral paths in the analyzer chamber. By scanning the radiofrequency or magnetic field, the ions can be detected sequentially.
2.5 Peptide sequencing and computer software
Tandem mass spectrometry (MS/MS) allows peptides to be sequenced without the need for complete separation,in contrast to Edman degradation. Furthermore, it allows for the rapid characterization of complex protein mixtures.
MS/MS experiments consist of two stages. First, a specific ion is isolated or selected, which is subsequently accelerated through a region of higher pressure containing collision gas (typically argon). In the second stage, the masses of the fragments are analyzed91,92.
Tandem mass spectrometry of peptides was first investigated by D. Hunt et al.93 and K. Biemann94.
The MS/MS spectra of peptides are relatively easy to interpret, since peptides typically break at predictable sites. Roepstorff and Fohlman95 developed a nomenclature for the ions observed, based on the site of cleavage. This
nomenclature is now generally accepted and is shown schematically in figure 6.
Mass specrometry for biomolecules
Figure 6. Nomenclature for peptides of Roepstorff and Fohlman.
Peptides are most likely to fragmentize at the amide bond, which is the weakest bond96. Dependent on the side that carries the charge, break-up results in y-ions or b-ions: y-ions for C-terminal ions and b-ions for N-terminal ions. Apart from these ions, a-ions are regularly observed. In tryptic peptides the N-terminus always consists of a basic lysine or arginine residue. As a result, y-ions are generally the most intense ions in mass spectra of tryptic peptides97.
Originally, mass spectra of peptides had to be interpreted manually. Subsequently, a homology search was performed in a database (using web-based database search programs such as BLAST or FASTA)98. Such an approach only allows for limited throughput.
Several software algorithms for automatic sequencing of MS/MS spectra have been developed in the late 1990s99. The one most widely used is Sequest100 which was developed by J. Yates and J. Eng at the University of Washington, Seattle.
The program initially only allowed for the searching of unmodified peptides, but later was extended to include the possibility of modified ones101.
This software searches for all peptides in the sequence database that have the same mass as the parent ion and subsequently match the theoretical MS/MS spectrum with the one that was found experimentally. Because of the enormous amount of data generated in a typical run, it is virtually impossible to interpret the MS/MS spectra manually. Computer software has become indispensable in studies of the proteome and was to a great extend responsible for the success of the shotgun approach.
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Chapter 3
Capillary electrophoresis
and capillary isoelectric focusing
3.1 Capillary electrophoresis and proteomics
In the last decade, the field of proteomics has grown immensely. Key to this research is the ability to separate a large number of proteins in parallel. The most widely used separation method in proteome analysis is 2D gel electrophoresis (2D- GE). Despite its unmatched resolution, the inability of 2D-GE to enable high throughput analysis gave rise to the need for alternative approaches.
One such alternative is the shotgun approach1,2 in which liquid chromatography (LC) is used to separate digests of protein mixtures. Protein identification is obtained by performing tandem mass spectrometry (MS/MS) of the individual peptides.
Although separation of peptides or proteins is commonly performed by LC, CE has some clear advantages3,4. CE is a versatile technique with high separation
efficiency. Its fast performance combined with low sample consumption has made CE-MS a valuable, complementary tool to the widely used LC-MS for the analysis of biomolecules5.
3.2 Development of capillary electrophoresis
Capillary electrophoresis is a technique in which charged analytes migrate through a capillary, driven by an electric field. Apart from their own electrophoretic
mobility the analyte ions are driven by a bulk flow, which is present due to electroosmosis6,7.
The principle of capillary electrophoresis was introduced by Hjerten8, who was the first to describe electrophoresis performed in a tube, in stead of the commonly used gel. This format provided several advantages, such as the possibility of on- line detection, improved separation efficiency and shorter analysis times. Hjerten used tubes of 1-3 mm for his experiments. However, the technique came to its full potential when Jörgenson and Lukacs described electrophoresis in tubes of 100 µm9-12. Nowadays, capillaries of 50-75 µm are commonly used.
The first applications focused on the separation of biomolecules. CE was
considered to be the extension of gel-electrophoresis, which was the standard tool for these compounds. Now, the most frequent use of CE is in the pharmaceutical field13, but also for the analysis of biomolecules CE remains an alternative for LC14. There are several advantages of CE over LC. These include the possibilities of reduced run times, operating costs and solvent consumption. Furthermore, it offers superior separation efficiency and the sample consumption is low. However, the technique is limited by poorer injection precision. Furthermore, because of its
Capillary electrophoresis and capillary isoelectric focusing small injection volumes and consequently low mass flow, the concentration
sensitivity of a CE-system is low when compared to LC13-15. 3.3 Principles of capillary electrophoresis
3.3.1 Principle of separation
In CE, separation is based on differences in electrophoretic mobility of different compounds under an applied electrical field16. The mobility (μ) is proportional to the electrical force and the charge of the analyte, but is counteracted by frictional forces. These frictional forces are dependent on the viscosity of the solution and the radius of the analyte. Roughly spoken, mobility is proportional to the size -to- charge ratio of the analytes. Semi-empirical and theoretical models have been described to predict mobility. Offord proposed the following formula in 1966 for the mobility of peptides, presuming spherical shapes17:
μ = k.Z.M –2/3
In this equation, k is a constant, Z represents charge and M represents mass. This relationship has been verified empirically by Rickard et al.18
However, the velocity of the analytes is not only determined by their intrinsic electrophoretic mobility: it is also dependent on the electroosmotic flow (EOF) inside the capillary. Fused silica capillaries contain silanol groups at the surface.
Deprotonation of the silanol groups of the fused-sillica capillary wall (with pKa
values of 3-5, dependent on the quality of the capillary production lot19,20) leads to an excess positive charge in the liquid phase. The positive ions form a double- layer near the inner surface of the capillary wall to balance its negative charge21. When voltage is applied, the positively charged counter-ions are drawn towards the cathode, together with their associated solvating water molecules. This results in the EOF, a flow of the liquid towards the cathode, which is constant along the whole capillary22,23.
The total mobility of an ion can now be seen as being constituted of two parts - its intrinsic mobility (μins) and the EOF24:
μtot = μins + μEOF
The EOF is both dependent on the diameter of the capillary used and on the pH of the buffer solution. At low pH values, the deprotonation of the capillary wall is limited, resulting in low flow rates. Likewise, higher pH values result in a higher EOF.
35
The most basic form of capillary electrophoresis is capillary zone electrophoresis (CZE), for which the basic principles have now been discussed. Specific demands for the analysis have led to the development of other modes of separation, like isotachophoresis (ITP) and capillary isoelectric focusing (CIEF).
3.3.2 Joule heating
Several factors affect zone broadening. These include diffusion, Joule heating, wall adsorption, and injection plug length25,26, which all will be discussed below.
During the electrophoretic separation, heat is generated because of the current running through the capillary: Joule heating. Joule heating can cause the formation of a temperature gradient inside the capillary, which results in band broadening and possibly even the generation of gas bubbles inside the capillary27. This limits the ionic strength (conductivity) of the separation buffers that can be applied successfully. Joule heating can be controlled by operating at a voltage where generated heat can be effectively dispersed. Its effect can also be
compensated by enhancing the surface-to-volume ratio, i.e. using capillaries with smaller diameter28-30. Furthermore, cooling may be applied to prevent Joule heating31.
3.3.3 Injection methods
To maintain high separation efficiency, the method of sample injection proves to be a vital step28. Sample should be delivered in small volumes into the capillary reproducibly32-34. The most common methods are electrokinetic injection and hydrodynamic injection.
For electrokinetic injection, the capillary inlet and the electrode are inserted in the sample vial. A voltage is applied for a brief period of time. Subsequently, the compounds migrate into the capillary, as a result from electrophoretic migration and EOF.
In this technique, compounds with higher mobility are biased. These will be injected in larger quantities than the less mobile compounds. Furthermore, for run-to-run reproducibility sample buffer should remain unaltered, since changes in ionic strength result in differences in electrophoretic mobility and electroosmotic flow35.
Hydrodynamic injection can be performed by gravity flow, pressure or vacuum. No bias exists for different compounds. Gravity flow is executed by inserting the capillary in the sample vial, which is lifted to a certain height (Δh) with respect to the outlet vial. For pressurized injection, a pressure is applied at the inlet vial, pushing sample into the capillary. Similarly for vacuum injection, vacuum is
