Peptide profiling by capillary separation techniques coupled to mass spectrometry

Citation

Gaspari, M. (2006, December 13). Peptide profiling by capillary separation techniques coupled to mass spectrometry. Retrieved from https://hdl.handle.net/1887/5431

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Chapter 1

Capillary chromatographic separations and MS

Mass spectrometry (MS) is giving an ever-increasing number of outstanding contributions to many areas of research, spanning from analytical chemistry to bio-medical sciences. It has also been recognised to play a key role in the developing field of proteomics1-3 , which has undoubtedly become one of the research mainstreams of this decade.

The power of MS has been frequently boosted by coupling it to a separation device at the front end, allowing the analysis of more complex analyte mixtures. Hyphenated techniques like liquid chromatography-mass spectrometry (LC-MS) or capillary electrophoresis-mass spectrometry (CE-MS) are often able to routinely profile hundreds to thousands of analytes in a single run. Thanks to the continuous increase of MS performance and to its ease of combination with miniaturised analytical separations, mass spectrometry is proving fitter and fitter to solve specific biological problems and advance discovery in the life sciences4-8.

1.1 Mass spectrometry

The first major advancement in modern biological mass spectrometry can be indicated as the discovery of two soft ionisation techniques: electrospray ionisation (ESI)9 and matrix-assisted laser desorption ionisation (MALDI)10. These allowed polar molecules and macromolecules to be available for mass spectrometric analysis by typically generating a single, intact gas-phase ion out of 1000-10000 molecules of starting analyte of interest.

The discovery of MALDI and ESI as exceptional tools to achieve ionisation of intact biomolecules stimulated research and development on the mass analysis and detection sides. Instrumental advancements mainly resulted in improved mass accuracy, sensitivity, dynamic range and scan speed of new-generation mass spectrometers. These improvements have been critical for the study of complex peptide, protein or metabolite mixtures.

1.1.1 Electrospray ionisation

already in 1968. The original idea was developed and put into practice fifteen years later by John Fenn and co-workers, who introduced electrospray as a ionisation technique for mass spectrometry in 19849. The success of the approach of Yamashita and Fenn was mostly due to critical adjustments to the electrospray experimental setup and to their choice of approaching electrospray analysis starting from the investigation of very small molecules ( < 400 Da). In fact, while gradually moving towards the analysis of higher Mw biomolecules, they discovered the phenomenon of multiple charging. This peculiar characteristic of electrospray ionisation allowed macromolecules several tens of thousands Daltons large to be measured with a quadrupole mass spectrometer having an upper limit of 1500 m/z. Demonstration of ESI-MS analysis for large macromolecules was first reported in 198812.

ESI-MS analysis generally consists in infusing a dilute analyte solution through a fused silica capillary which, at its outlet, is inserted into a metal needle held at high electrostatic potential (3-5 kV), either negative or positive, relative to the MS inlet. The electrical field causes the analyte solution to extend towards the counterelectrode, ultimately generating a fine mist of charged droplets (called Taylor cone) as soon as the applied voltage exceeds a specific threshold value which depends on various experimental parameters (solvent flow rate and surface tension, needle dimension, distance from the counterelectrode, etc.).

The exact mechanism of ion formation from the charged droplets generated during ESI has been extensively debated in the literature. Two models have survived falsification, without replacing one another but complementing each other. The first is the coulomb fission mechanism, which assumes that a charge density limit is reached on the surface of the charged droplets due to evaporation. This surface charge density “overload” would be the driving force for droplet division into smaller droplets to increase surface area. Repetitive coulombic “explosions” and droplet shrinkage (due to solvent evaporation) would ultimately lead to a single ion.

1.1.2 Nanoelectrospray ionisation

In the early nineties, three separate groups reported exciting new directions in electrospray, which were provided by the miniaturisation of the electrospray interface itself. Gale and Smith15 first described the use of small-bore fused silica capillaries as ion source. Capillaries down to 5 μm internal diameter (ID) were etched in hydrofluoric acid to generate a sharp tip, and used as spraying needles for ESI ionisation of several biopolymers. One year later, Emmet and Caprioli16 reported sensitivities in the attomole range by integrating a chromatographic packing into the tip of an electrospray needle. Peptides bound to the stationary phase were stepwise eluted at sub-microliter/min flow rates. The third and probably most important contribution to the development of the nanoelectrospray ionisation interface came from the work of Wilm and Mann. First, they reported a theoretical description of electrostatic dispersion in electrospray17, which allowed them to quantitatively describe phenomena like the electrospray onset voltage, and the diameter of the droplets ejected by the Taylor cone. Their theoretical model led them to designing an electrospray interface optimised in terms of efficiency of ion production. In a following contribution18, they extensively reported the analytical properties of the newly developed source, which they named nanoelectrospray (nanoES) because of the low nL/min flow rates of operation and the size of the ejected droplets in the nanometer range. The contribution of this last group has been of special interest because of the theoretical description of many parameters affecting the electrospray process at normal and low flow rates. In addition, the first theoretical paper was followed by several applications to protein and peptide analysis of outstanding importance for biological research19, 20.

Mechanism of nanoelectrospray

in a fine mist of very small, charged droplets, from which charged analytes are formed by any of the proposed electrospray mechanisms described above.

+ +1-2 kV HV power supply + - electrons electrons Conductive coating 10-20 µm MS inlet + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Fused silica Taylor cone

Infusion solution ++ +1-2 kV HV power supply + -HV power supply + HV power supply + - electrons electrons Conductive coating 10-20 µm MS inlet + + + + + + + + ++ + + + + ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Fused silica Taylor cone

Infusion solution

Figure 1 Schematic of the nanoelectrospray interface. Operation in the positive ion mode is illustrated.

The onset voltage has been theoretically calculated by Wilm and Mann17. The equation they derived is:

Ut = 0.863(γr1/ε0)0.5

Where Ut is the onset voltage, γ is the surface tension of the solution being sprayed

and r1 is the distance between the tip and the counterelectrode. Experimental results

were very well in agreement with the theoretical model of Wilm and Mann, but only for small distances from the counterelectrode (1-2 mm). At larger distances, the slope of the curve correlating the onset voltage and the square root of the electrode distance r1 was lower than predicted. This phenomenon is readily observable in most nanoES

In the theoretical description of Wilm and Mann, a calculation of the size of the droplets emitted by the Taylor cone was also reported:

ρ 4π2_{γ tan} π _φ 2 r_e= U_a U_t 1 dV dt 2/3 ρ 4π2_{γ tan} π _φ 2 r_e= r_e= U_a U_t 1 dV dt 2/3

Where re is the emission region radius from which droplets are ejected, γ is the surface

tension of the liquid, φ the liquid cone angle, ρ the density of the liquid, Ut the

threshold voltage, Ua the applied voltage and dV/dt the flow rate.

The most important part of this equation is the term (dV/dt)2/3 which describes that the droplet emission region radius is proportional to the 2/3-power of the flow rate. In other words, a 100-fold decrease in flow rate would theoretically yield a (100)2/3-fold decrease in droplet radius, meaning a (100)2-fold decrease in droplet volume. This ultimately means that, while normal electrospray interfaces yield droplets containing tens of thousands of analytes per droplet (assuming moderate analyte concentrations), droplets generated by nanoESI only contain an average of a few analytes/droplet, more readily available for ionisation. By reasoning that a smaller droplet diameter would have beneficial characteristics in terms of ion formation from charged droplets, the researchers were pushed to lower the flow rate of their electrospray interface as much as they could.

Their nanoES interface gave a ionisation yield of 1 in 390 molecules for a synthetic peptide. This efficiency was reported to be several hundreds-fold higher than what could be obtained by conventional ESI interfaces.

Characteristics of the nanoelectrospray interface

guaranteed high sensitivity and very long acquisition times for tandem mass spectrometry (MS/MS) experiments, often necessary for protein identification. Furthermore, the nanoES interface has proved to be more tolerant to salts and contaminants compared to the normal ESI interface. In fact, the very challenging direct MS analysis from minimal amounts of crude biological samples has been demonstrated21.

An extremely important characteristic of the nanoES interface which perhaps has only

tion process:

.1.3 Mass analysis

t mass analysers is to separate charged analytes in time or in

s in space. A specific sinusoidal potential is applied to an array of four cylindrical or hyperbolic rods recently been gaining the deserved attention is the reduced risk of ion-suppression effects. In normal ESI, fissioning is often involved in the process of ionisation. Fissioning refers to a series of consecutive Coulombic explosions that an ESI-generated droplet undergoes before releasing a single gas-phase ion. Fissioning favours ionisation of surface-active analytes, i.e. analytes highly present at the droplet surface, which are more likely to concentrate in the offspring droplets22 and increase their chance of ionisation. This bias towards surface-active analytes strongly decreases the dynamic range of standard ESI ionisation. This is of course an important drawback, especially in case of the analysis of highly complex samples containing analytes at concentration levels differing by several orders of magnitude.

The nature of nanoelectrospray, instead, favours a more unbiased ionisa

the droplets created are so small that gas-phase ions can often be produced directly from them23, without relying on fissioning. Nanoelectrospray is thus more suitable for the analysis of molecules with reported low surface activity and, in general, for the analysis of highly complex mixtures.

1

The objective of mos

space before ion energy is converted to electrical impulse at the MS detector. The only notable exception to this concept is the one on which Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) is based. In fact, FTICR MS is a non-destructive type of mass measurement, as it will be discussed below. Mass analysers used today in combination with soft ionisation techniques like ESI are of many kinds, offering a wide range of performance in terms of mass resolution, mass accuracy, sensitivity and dynamic range24. There are four basic types of mass analyser: quadrupole (Q), ion trap (IT), time-of-flight (TOF) and FTICR.

constituting the quadrupole. The electric field generated only allows ions with a specific m/z to pass through. Mass scanning is obtained by changing the sinusoidal potential, thus allowing ions at different m/z to reach the detector. A more sophisticated version of the quadrupole mass analyser is the triple quadrupole (QQQ). This configuration allows tandem mass spectrometry experiments (MS/MS) to be performed. Tandem mass spectrometry in QQQ instruments consists in selecting an ion at a particular m/z in the first quadrupole, fragmenting it through impacts with a collision gas in the second quadrupole (Q2), and generating a mass spectrum of the fragments produced in Q2 with the third quadrupole. MS/MS can provide tremendous improvements in terms of signal-to-noise ratio (S/N) and specificity. Very specific and sensitive MS/MS scanning modes, together with the great dynamic range provided by Q-based mass spectrometers, still make the triple quadrupole the most powerful solution for MS-based quantitative analysis.

Quadrupole ion traps (IT), instead, separate ions by first confining them in a small region of the mass spectrometer, the “trap”, and then sequentially ejecting them out

ated by an electric field, which imparts the same kinetic energy to all towards the detector according to their m/z value. In the three-dimensional ion traps, the trapping action is made possible by three cylindrically symmetrical electrodes: two end-cap electrodes held at ground potential and a ring electrode on which a radio frequency voltage (RF) is applied. After trapping, the ions are sent to the detector by scanning the RF field to higher values, thus creating instable trajectories for ions of increasing m/z values, which are sequentially ejected out through small holes in one endcap electrode (Figure 2). Due to their relative low cost, sensitivity and multi-stage MS/MS capability (MSn), ion traps have been the workhorse of MS-based proteomics to date. Their excellent sensitivity is due to both their compact size, which enhances ion transmission, and their high duty cycle (the number of ions that the analyser is able to send to the detector divided by the number of ions that actually enter the mass spectrometer).

TOF analysers work on a simple principle. A package of ions generated in the ion source is acceler

Description of the three-dimensional ion trap cell. Ring electrode Ring electrode Endcap electrode Endcap electrode Trapping region Ion entrance Ion exit To detector From source RF voltage voltage RF Ring electrode Ring electrode Endcap electrode Endcap electrode Trapping region Ion entrance Ion exit To detector From source RF voltage voltage RF Figure 2

pace nor in time. Their m/z ratio is alculated by monitoring their motion around the axis of a magnetic field. The

ew engineering or on the coupling of more than one In FTICR analysers, ions are separated neither in s

c

operating principle of the spectrometer is rather complex. Briefly, ions are trapped in a magnetic field generated by a superconducting magnet. Their orbiting motion induces a flow of electrons in a circuit outside the ICR cell. This flow of electrons generates a detectable current called image current after a RF excitation is applied to the trapped ions. The image current can be resolved in its frequency components by using Fourier transform. The frequency components, calculated with extreme accuracy, allow for the calculation of very precise and accurate m/z values of the ions generating them. Ion detection in FTICR MS is often appropriately indicated as listening to the circulating

ions. FTICR is the high-end performing mass spectrometer. Newly commercialised

instruments (see below) promise to allow access to high MS performance to a broader research community. This could ultimately have a tremendous impact on research in biological mass spectrometry.

The last years have seen the commercialisation of new, more powerful mass spectrometers based either on n

MALDI-TOF/TOF spectrometers provide excellent sensitivity and throughput for protein identification, giving the possibility of re-analysing the prepared sample multiple times, which is particularly desirable in mapping complex protein mixtures or post-translational modifications. Though commercially available fraction collectors are now allowing the coupling of nanoscale separations to MALDI-TOF/TOF mass spectrometers, complex peptide mixture analysis requiring nanoLC-MS are still preferably performed on mass spectrometers based on ESI/nanoESI interfacing. Among these, it is worth mentioning two recent advances. The first is the two-dimensional linear ion trap (LT)26, which provides a much larger trapping volume, thus boosting the ion trap performance from the point of view of sensitivity and dynamic range. Both sensitivity and dynamic range were in fact limited in the small three-dimensional traps by the phenomenon of space charging, i.e. interferences in the applied electric field caused by the accumulation of an excess of ions in a confined space. The second, impressive new advancement is the hybrid LT-FTICR mass spectrometer27, which utilises a linear trap as ion storage device in front of a highly-performing FTICR mass analyser. This allows an easy control of the ICR trap-filling step and allows several parallel MS scanning modes of the two mass analysers. In the most used scanning mode in peptide analysis, the two MS analysers are in fact operated in parallel: while a high accuracy FTICR scan is performed in 1-2 sec. time, fast MS/MS acquisition on the linear trap allows several analytes to be fragmented for identification. The combined information of high mass accuracy on the parent ion with MS/MS information is greatly beneficial for peptide identification experiments.

1.2 Capillary chromatographic separations

ytical separations in order to expand eir role within life science research. Chromatographic-based separations performed

erformance of analytical separations in terms Miniaturisation has been a necessary step for anal

th

in fused silica capillaries or in etched microchannels have been crucial for solving problems in sample-limited situations.

The extreme complexity of analytical problems routinely encountered in life science research calls for a further increase in p

additional separation dimension, analytical separations coupled to mass spectrometry can thus be able to resolve hundreds to thousands of components in a single run.

1.2.1 Nanoscale liquid chromatography (nanoLC)

been the workhorse of protein

red an important role

ing High performance liquid chromatography (HPLC) has

and peptide separation since its development in the 1970`s. HPLC columns have been usually produced by packing millimeter-bore columns with silica particles having diameters smaller than 10 μm and porosity between 60 and 300 Å. The most popular separation mode for proteins and peptides has probably been reversed phase (RP) chromatography. This mode is based on partition between a polar mobile phase and an apolar stationary phase obtained by derivatisation of the silica surface by alkyl chains having lengths spanning from 4 to 18 carbon atoms (C4 to C18). Chromatographic

modes based o ionic interactions, namely anion exchange (AEX) and cation exchange (CEX) chromatography have also been widely used in peptide and protein analyis. AEX and CEX are based on derivatisation of the silica surface with, respectively, a basic or an acidic group. In ion exchange chromatography, peptides and proteins bound to the stationary phase via ionic interactions are subsequently eluted from the column by a mobile phase gradient of increasing ionic strength.

RP and ion exchange chromatography (IEX) have quickly conque

The gain in absolute sensitivity that can be achieved by downscaling an LC-MS separation can be indicated by a sensitivity factor29:

d

f ~

d

d

f ~

d

Where d1 and d2 are the inner diameters of the larger and the smaller column,

respectively. It can be easily calculated that moving from a separation performed in a 2 mm ID column, a standard format for LC-MS analysis, to a nanoscale separation in a 75 μm ID column, the theoretical concentration factor is about 1000.

Since its early reports, nanoLC-MS soon demonstrated femtomole sensitivity in peptide analysis30, and the ability to deal with complex mixtures of digested proteins. Applications of nanoLC-MS in proteomics will be discussed in chapter 2.

1.2.2 Capillary electrochromatography

(where only the capillary walls generate EOF) or monolith-CEC. High efficiency in CEC is mainly due to the flat flow profile generated by EOF as opposed to pressure-driven flows proper of HPLC, where the liquid is forced from one end of the column to the other, generating a parabolic flow profile responsible for a positive contribution to plate height. This ultimately limits the efficiency obtainable with pressure-driven flow techniques. The flat flow profile generated by EOF is due to the fact that the driving force is uniformly applied to the bulk solution at every point inside the capillary.

-+

Fused silica capillary

Silica particles - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - --_- -- --- --_- -- -- - --_- -- --- --_- -- -- --_- -- --- --_- -- -- - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - --_- -- --- --_- -- -- - --_- -- --- --_- -- -- --_- -- --- --_- -- -- - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - --_- -- --- --_- -- -- - --_- -- --- --_- -- -- --_- -- --- --_- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- - --_- -- --- --_- -- -+ + _{= solvated proton} + + + + + + + + + + + + + + + +++ + + + + + + + + + + + + + + + + + + + + + + + + + + + +++ + + + + + + + + + + + + + + + + + + + + + + + + + + + +++ + + + + + + + + + + + + + + + + + + + + + +++ + + + +++ + + + + + + + + + + + + +++ + + + +++ + + + + + + + + + + + + +++ + + + + + + + + + + + + + + + +++ + + + + + + +++ + + + + + + +++ +++ + + + + + + + + +

-Figure 3 Electroosmotic flow in packed-CEC columns.

As opposed to CE, separation in CEC can be influenced both by the analyte’s electrophoretic mobility and by its partition coefficient with the stationary phase. Thus, CEC can rely on a wide panel of stationary phases (often borrowed from classical chromatography) in order to tune selectivity, resulting in a more flexible separation tool compared to CE.

eliminates the need for a retaining frit, thus minimizing air bubble formation, while the high permeability of the media allow for high electric fields to be applied for separation. Research efforts are being made to transfer the monolithic concept in a chip format33.

Though HPLC and nanoscale HPLC still rule in peptide separation, peak capacities required in complex profiling applications in the field of proteomics call for more performance. CEC separations, especially if performed in chip-based format, might become a valid alternative to nanoscale HPLC if they will prove to be able to deliver robust performance in real-life applications.

1.3 Interfacing separations by nanoES ionisation

Even with the improvements that nanoES has brought in terms of minimising ion suppression effects, still a front-end separation before MS detection is necessary in the analysis of many biological samples. The new generation of “omics” approaches, metabolomics and proteomics analyses, aim at detecting at least thousands of analytes present at very different concentration levels in a single sample. Current MS instrumentation has neither sufficient resolution nor the required dynamic range to perform these analyses alone. New FTICR-based mass spectrometers27 hold new promises in this respect, but their capability of handling complex biological samples without any chromatographic separation still has to be demonstrated. For now, “omics” analyses by mass spectrometry are very often performed in combination with some sort of chromatographic separation, achieved in capillary format in case of sample-limited situations.

+1-2 kV Conductive coating

From liquid

separation Nanoflow union

Nanospray

…..

+1-2 kV Conductive coating

From liquid

separation Nanoflow union

Nanospray

….. …..

Figure 4Nanoelectrospray interface based on conductively coated tips.

A second approach makes use of a remote contact, and thus consists in having the high voltage applied far from the tip outlet, as shown in Figure 5. The capillary carrying the liquid separation is connected to a spraying tip via a conductive nanoflow union, or, alternatively, a tee can be used, where the third port is connected to a conductive Pt wire used for applying the voltage. Usually, the distance between the remote contact and the tip outlet can vary from 1 to 10 cm. This approach is definitely more robust than the previous one, because no deterioration in electrospray performance is observed in the short term, opposite to the conductively coated capillaries. System non-stop operation can last 2-3 weeks. Tip clogging is the most important cause of system failure after long operation times.

Nanospray ….. From liquid separation +1-2 kV Pt wire Nanospray ….. ….. From liquid separation +1-2 kV Pt wire

Figure 5 Nanoelectrospray interface based on remote contact.

An important difference between the two types of nanoES interfaces here described concerns their electrochemistry. In general, all electrospray interfaces can be seen as closed circuits, where excess of charge is produced at the electrospray tip, causing the formation of a mist of charged droplets. This excess of charge requires electrochemical reactions to occur at the interface between the conductive metal contact and the solution being sprayed. For example, positive ion mode electrospray generally produces Fe2+ ions from oxidation of the stainless steel contact37. With conductively coated nanoES tips, electrochemical reactions occur at the tip outlet, so excess charge is generated at the site of electrospray. With a remote contact, instead, the excess charge generated has to be conducted from the site of creation to the site where electrospray takes place, a few cm further. This can be accomplished by a convective flow of the effluent itself, but also requires a sufficiently conductive solution to help transporting the excess charge.

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