MALDI-TOF: a powerful tool for the analysis of biomolecules and large organic molecules

III Workshop 2013-2014 Corso del Dottorato in Ingegneria dei Materiali del Politecnico di Milano Methods of Characterization of Materials MALDI-TOF: a powerful tool for the analysis of biomolecules and large organic molecules Anna Paola Caricato Department of Mathematics and Physics E. De Giorgi University of Salento Lecce, Italy Milano 22 October 2014

Outline Mass Spectrometry: MALDI: - overview of the basic principles and instrumentation - overview of the basic principles; - experimental set-up; - sample preparation; - main requirements; - the physical process; - MALDI vs ESI; - applications; CONCLUSIONS

Mass spectrometry? Gas Phase Sample + _ Ionizer Mass Analyzer Detector It is an analytical technique that helps to identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions

Mass spectrometry (MS)? Gas Phase Sample + _ Ionizer Mass Analyzer Detector MS permits to: - identify unknown compounds; - determine the isotopic composition of elements in a molecule - determine the structure of a compound by observing its fragmentation; - quantify the amount of a compound in a sample; - study the fundamentals of gas phase ion chemistry

Mass spectrometry? Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. m/z ratio

Mass spectrometry: important performance factors Mass accuracy : How accurate is the mass measurement? Resolution : How well separated are the peaks from each other? Sensitivity : How small an amount can be detected / analyzed?

Mass spectrometry: Important performance factors In mass spectrometers, two peaks are considered resolved if two adjacent peaks with equal intensity are such that the height of the valley is 10% of the higher peak The resolving power R is the capability of distinguish two peaks of different mass: R = Where m 2 is the higher m/z value High resolution means R > 5000 Low resolution means R < 5000 m2 m m 2 1

Mass spectrometry: Important performance factors Increasing the resolving power increases the accuracy which indicates how much the measured mass is different by the real mass. It is the error in the measure!! The accuracy is defined as 1/R Accuracy = m m m 2 1 6 10 2 To indicate the error in ppm

Mass spectrometry: Important performance factors

Mass spectrometry: Important performance factors High resolution means better mass accuracy 8000 15 ppm error Resolution =18100 Counts 6000 4000 24 ppm error Resolution = 14200 2000 55 ppm error Resolution = 4500 0 2840 2845 2850 2855 Mass (m/z)

Mass spectrometry: principle of the technique High Vacuum System 10-4 Pa Inlet Ion Source Mass Analyzer Detector Data System m/z ratio

Mass spectrometry: principle of the technique High Vacuum System 10-4 Pa Inlet Ion Source Mass Analyzer Detector Data System - Electronic Impact (EI); - Chemical Ionization (CI); - MALDI; - ESI

Mass spectrometry: principle of the technique Ionization mechanisms e - expulsion: M M +. + e - Protonation: M + H + MH + Cationization: M + Cat + MCat + deprotonation: MH M - + H +

Electron Impact Ionization In an EI ion source, electrons are produced through thermoionic emission. The electrons are accelerated to 70 ev. HARD technique ionization and fragmentation The radical cation products are then directed towards the mass analyzer by a repeller electrode

Electron Impact Ionization

Chemical Ionization It is based on the chemical reaction of the sample with an ionized reagent Less fragmentation, and simpler spectrum respect to EI

Electro Spray Ionization It produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized.

Mass spectrometry: principle of the technique High Vacuum System 10-4 Pa Inlet Ion Source Mass Analyzer Detector Data System - Quadrupole - Magnetic Sector - Time of flight (TOF) - Ion Trap

Mass analyzer: Quadrupole An oscillating potential is applied to the bars Only for paricular value of the RF and of the Voltage, ions with a particular m/z ratio can go out from the bars to the detectors performing a sinusoidal motion. The others will not be able to flow between the bars and will crash on some device elements and will not be detected 20 cm m/z V changing V and RF a scan over the masses is possible Resolving power = 3000

Mass analyzer: Magnetic Sector Kinetic energy of the ions zv = ½ mv 2 Force due to B: Bvz = mv 2 r Combining the two equations: m z = 2 B r 2V 2 Detector For B and V only the ions having the right mass will go out from the analyzer

Mass analyzer: time of flight Principle : If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass-to-charge ratios.

Mass analyzer: time of flight L Ion kinetic energy From cinematic v=l/t

Mass analyzer: time of flight The mass-to-charge ratio of an ion is proportional to the square of its drift time. m/z is strictly connected to the time of flight t = Drift time L = Drift length m = Mass V = Voltage z = Number of charges on ion Small ions reach the detector before large ones

Mass analyzer: time of flight (TOF) Can we compensate for the initial energy spread of ions of the same mass to produce narrower peaks? Delayed Extraction Reflector TOF Mass Analyzer

TOF + Delayed Extraction (DE) improves performance 0 V. + + + Ions of same mass, different velocities Step 1: No applied electric field. Ions spread out. 0 V. 20 kv. + + + 0 V. Step 2: Field applied. Slow ions accelerated more than fast ones. 20 kv. Step 3: Slow ions catch up with faster ones. + + + 0 V.

TOF + Reflector A single stage gridded ion mirror that subjects the ions to a uniform repulsive electric field to reflect them. Detector Ion Source Reflector (Ion Mirror) The reflector or ion mirror compensates for the initial energy spread of ions of the same mass coming from the ion source, and improves resolution.

TOF + Reflector In this way the resolving power is > 10 4 Figure from: Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ. 1997, 74, 1289.

TOF + Reflector

Mass Analyzers: comparison

Mass spectrometry: principle of the technique High Vacuum System 10-4 Pa Inlet Ion Source Mass Analyzer Detector Data System - Electron multiplier - Microchannel plate Photomultiplier

Mass Spectrometry: detector An electron multiplier is a vacuum-tube structure that multiplies incident charges. In a process called secondary emission, a single electron can, when bombarded on secondary emissive material, induce emission of roughly 1 to 3 electron. If an electric potential is applied between this metal plate and yet another, the emitted electrons will accelerate to the next metal plate and induce secondary emission of still more electrons. This can be repeated a number of times, resulting in a large shower of electrons all collected by a metal anode, all having been triggered by just one.

Mass spectrometry: principle of the technique High Vacuum System 10-4 Pa Inlet Ion Source Mass Analyzer Detector Data System

MALDI: Matrix assisted laser desorption ionization Mass Spectrometry (MS) Vital tool used to characterize and analyze molecules Limitations Biomolecules and organic macromolecules are fragile Molecular ions or meaningful fragments were limited to only 5-10 kda at the time 1987 Introduction of a new laser-based technique MALDI: Matrix assisted laser desorption ionization

MALDI: Principle MALDI is a soft ionization technique that allows the desorption and ionization of large molecular species ~ 10 1000 kda MALDI is based on the discovery that dissolving a biomolecule (like enzyme, proteins, DNA, e.t.c.) within a great excess of a particular matrix (usually 2,5-DHBdihydroxybenzoic acid) specifically chosen to absorb at the irradiation wavelength can lead to its ejection into the gas phase and ionization with MINIMUM DEGRADATION

MALDI: Principle When used in combination with a mass spectrometry (MALDI-MS) it allows the analysis of biomolecules and large organic molecules increasing the sensitivity of conventional mass spectrometry instruments (100 /240 Da).

MALDI: Principle In 1988 at the International Mass Spectrometry Conference in Bordeaux the first MALDI spectrum of a protein was presented Spectra of b-d-galactosidase. Matrix:nicotinic acid; Wavelength: 266 nm few weeks later Fast progresses!

MALDI: Principle

MALDI: Matrix Assisted Laser Desorption Ionization < 10 min

MALDI: Matrix Assisted Laser Desorption Ionization Sample plate hν Laser 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate. AH + 2. Laser flash ionizes matrix molecules. 3. Sample molecules are ionized by proton transfer from matrix: MH + + A M + AH +. +20 kv Variable Ground Grid Grid

MALDI: Principle Different steps take place in the MALDI process: 1. Sample preparation (dilution of the analyte molecules in a matrix with particular properties); A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added*. The matrix solution is mixed with the analyte (e.g. protein-sample). * The role of water and organic solvent allows both hydrophobic and hydrophilic molecules to dissolve into the solution

MALDI: Principle 2. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte molecules embedded into MALDI crystals. The matrix and the analyte are said to be co-crystallized.

MALDI: Principle 3. Excitation, by means of a UV (or IR) laser beam, of the sample and disintegration of the condensed phase; 4. Generation and separation of charges and ionization (protonation or deprotonation) of analyte molecules;

MALDI: Principle 5. Extraction and separation according to mass-to-charge ratio of the ions in the mass spectrometry; 6. Detection.

MALDI: Principle Despite the involvement of a very low absolute amount of material, a dense plume of material containing matrix neutrals as well as reactive species, such as matrix radicals, electrons, hydrogen atoms, is formed which is expanding into the vacuum of the mass spectrometer ion source. Within this plume regardless of a low average degree of ionization, suitable analytes can be ionized with very high efficiency and thus detected with high sensitivity.

MALDI: hardware Peaks are inherently broad in MALDI-TOF spectra (poor mass resolution). The cause: Ions of the same mass coming from the target have different speeds. This is due to uneven energy distribution when the ions are formed by the laser pulse. Sample + matrix on target Ions of same mass, different velocities + + +

MALDI: hardware Sample plate Extraction grids Prism Attenuator Laser Timed ion selector Reflector detector Reflector Linear detector Collision cell Pumping Camera Pumping

MALDI: hardware

MALDI: hardware (energy deposition is achieved via absorption by vibrational modes of the matrix molecules) O-H bending and C-H stretch vibrations: 10 µm C=O stretch vibrations: between 5.5 and 6.5 µm O-H or N-H stretch vibrations : ~ 3 µm

MALDI: Sample preparation The Matrix Contains ring structures to absorb energy from UV laser Contains acid group to protonate the analyte OH HOOC Matrix solution DHB or CHCA dissolved in acetonitrile and trifluoroacetic acid OH 2,5-dihydroxybenzoic acid COOH HO CN Sinapinic acid α-cyano-4-hydroxycinnamic acid

MALDI: Sample preparation There are many different matrices that can be used for MALDI - TOF. Some of the most common include:

MALDI: Sample preparation MALDI TOF spectrum of myoglobine Matrix: 2,5 DHB

MALDI: Main requirements The technique is mainly empirical and it is based on a trial and error approach (although, recently, molecular dynamic simulations have given a big help in understanding the different processes involved) on finding appropriate molecular matrices for the various types of biomolecules. However it is well recognized that A successful matrix should exhibit the following criteria: It has to isolate analyte molecules by dilution within the preparation, to prevent analyte aggregation. Moreover the exact dilution of the analyte is such that to reduce its thermal degradation. The critical analyte/matrix ratio increases with molecular weight (for peptide in the low kilodatlton range it is of about (1-5) 10-2 )

MALDI: Main requirements It has to absorb the laser energy* via electronic (UV-MALDI) or vibrational excitation (IR-MALDI) in order to have disintegration of the condensed phase without excessive destructive heating of the embedded analyte molecules. It has to be acidic in order to act as a proton source to encourage ionization of the analyte. At present, it is assumed that the matrix plays an important role in the protonation process Moreover for what about the laser beam Fast excitation is necessary in order to avoid destructive thermal excitation of the analyte: laser pulse duration 0.5 ns 10 ns (maximum 25 ns)

MALDI: Main laser irradiation parameters List of most relevant irradiation parameters* * K. Dreisewerd Chem. Rev. 103, 395-425 (2003)

MALDI: Main characteristics Presence of a threshold fluence for ion detection Strong dependence of the threshold fluence by the laser spot dimension Phase transition model: probably it does not exist a unique model. It depends on the amount of laser energy deposited The ion signal increase with the laser fluence (depending on the kind of matrix) Mean ion velocity: 200 1000 m/s Mean neutral velocity: ~ 500 m/s Ion/neutral ratio ~ 10-5 10-3

MALDI: the physical process At the first a three step model was supposed: - Incorporation and isolation of analyte molecules in the host matrix; - Their release into the vacuum upon disintegration of the matrix-analyte solid after laser-energy deposition; - Their ionization by ion-molecule reactions in the laser plume including matrix reactive ionic species this three-step model was not detailed enough to explain many of the striking characteristic features of MALDI The key problem in investigating MALDI is that it is a complex chemical event (onelaser-shot-one-masss pectrum); it is, moreover, happening within the time scale of a few nanoseconds.

MALDI: the physical process With the advent of delayed-extraction MALDI-TOF instruments not only the practical performance of MALDI-TOF instruments was substantially improved, but also a fundamental property of the MALDI ions became experimentally accessible, i.e., their average initial velocity. This initial ion velocity became an extremely valuable tool to characterize MALDI processes Characteristic for MALDI of peptides and proteins is a matrix-dependent high initial ion velocity of the analyte ions, typically between 200 and 1000 m/s.

MALDI: the physical process This initial ion velocity is in a first approximation independent of - analyte mass; - charge state and ion polarity. It is also not affected by the laser fluence within the analytically useful fluence range. Only those experimental variables affecting crystallization, such as solvents and additives, may result in a significant shift of v 0, with both directions occurring (some typical values of initial ion velocities are depicted in Table 1). M. Karas, R. Kru ger, Chem. Rev. 103 (2003)

MALDI: the physical process The high initial velocity shows a considerable spread (v 0 ± 0.5v 0 ) which is responsible for limitations in linear TOF analysis and can be further compensated for in reflectron configurations. The axial initial velocity is considerably larger than the radial velocity, pointing to a strongly forwarded orientation of the MALDI plume.

MALDI: the physical process This, together with the numerical simulations performed by Zhigilei and Garrison*, lead to the new picture that the generation and emission of matrix clusters is the fundamental ablation phenomenom. This cluster emission is accompanied by neutral evaporation, already occurring at lower laser fluences *Zhigilei, L. V.; Kodali, P. B. S.; Garrison, B. J. Chem. Phys. Lett. 1997, 276, 269. - Zhigilei, L. V.; Kodali, P. B. S.; Garrison, B. J. J. Phys. Chem. B 1997, 101, 2028. - Zhigilei, L. V.; Garrison, B. J. Rapid Commun. Mass Spectrom. 1998, 12, 1273. - Zhigilei, L. V.; Garrison, B. J. J. Appl. Phys. 2000, 88, 1281.

MALDI: the physical process The threshold behavior of MALDI ionization is now regarded as the critical point when the accumulated excitational energy density in the matrix solid reaches a critical value to induce an explosive cluster emission, and ionization is consequently linked to the occurrence of these clusters. Evaporational cooling within the decaying clusters is the reason for the softness of the MALDI process. The most straightforward means to generate charged species within a cluster ablation process is the statistical formation of clusters having a deficit/excess of ions; ionization is accomplished by charge separation. The energy required to overcome ion-pair interactions will be easily supplied by the mechanical energy of exploding clusters and the energy requirements may be further reduced by residual solvent The other possibility is by photoinization

MALDI: the physical process The final step of the ionization process Once charged cluster are produced, formation of final ions is accomplished by desolvation of matrix and or residual solvent: the energetically favorable proton-transfer neutralization within the clusters and the consecutive evaporation of the neutral species

MALDI: adduct ions Formation of MALDI adduct ions For analytes with high affinity with cations [M+Na] +, [M+K] + adducts can be formed

MALDI: Charge state

MALDI: Charge state MALDI descourages the formation of multiplycharged ions because:

MALDI: some spectra MALDI-TOF mass spectrum Peptide does not fragment 2683 Single pronotation state Soft ionisation 1343 double pronotation state Maldi TOF/TOF mass spectroscopic spectre for UCH-L3 digested Ecotin-Ubiquitin- FLS in the m/z range 799 4013. The peaks at 2683 and 1342 represent the single and double protonation states of the FLS peptide, respectively.

MALDI: some useful references For the desorption mechanism using UV laser (which means excitation of samples, the subsequent phase change, and the dynamics of the material plume expansion) look at the reference: K. Dreisewerd, Chem. Rev. 103, 395-425 (2003) Primary (matrix) and secondary (analyte) excitation and ionization mechanisms are reviewed in the two closely related articles by Karas and Kru ger** and Knochenmuss and Zenobi*** ** M. Karas, R. Kru ger, Chem. Rev. 103 (2003) ***R. Knochenmuss, R. Zenobi, Chem. Rev. 103 (2003)

MALDI: some useful references A review about IR-MALDI K. Dreisewerd, S. Berenkamp, A. Leisner, A. Rohlfing, C. Menzel, Int. J. Mass Spetrom. (2003)

MALDI: advantages-disadvantages DISADVANTAGES: the analysis of small molecules is still a problem ADVANTAGES: 1. The necessary sample preparation is simple and fast; 2. Tolerance to the impurity presence as salt or buffer contaminations; 3. Presence of nearly exclusive singly charged ions; 4. Minimum time of acquisition (200 samples analysed in 1 h); 5. Extreme sensitivity: even single cells may be analyzed and it has also been demonstrated that already 10 molecules (ca. 700 yoctomoles) of an analyte are sufficient to give a detectable signal limits of modern MS detectors.

MALDI vs ESI Sample MALDI-TOF Solution but ends up embedded in crystalline matrix Sample tolerant to salts Singly charged ions ESI Solution eg can come straight from HPLC Results can be affected by salts eg phosphates Multiply-charged ions Adduct-formation not so Common. Appearance one peak Adducts with salts common May be difficult to interpret spectra Appearance many peaks due to multiple charges

MALDI: spectrum identification Different softwares are available for spectra analysis

MALDI: spectrum identification Free database for protein analysis http://prospector.ucsf.edu/prospector/mshome.htm http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msfitstandard

MALDI: spectrum identification

MALDI: fields of applications

MALDI: fields of applications - Molecular weight Screening; - Protein Identification; - DNA and RNA sequence analysis; - In-source fast fragmentation; - Analysis of Polymer and Polymer Blends; - Clinical Microbiology; - Clinical chemistry; -...and many others

MALDI: some spectra 40000 MALDI TOF spectrum of IgG MH + Relative Abundance 30000 20000 (M+2H) 2+ 10000 (M+3H) 3+ 0 50000 100000 150000 200000 Mass (m/z)

MALDI: some spectra

MALDI: some spectra The direct analysis of viruses, bacteria, fungi and spores is possible. Protein composition differ between bacterial specied (even between bacterial strain), different spectra will be generated, allowing discrimination between closely related organism. In the case of bacterial or fungal identification, a microbial colony is analyzed, or in some cases, direct blood culture material, urine, cerebrospinal fluid.

MALDI: some spectra Maldi-Tof spectra of intact B cereus T spores Matrix: sinapinic acid *C. Fenselau et al. Mass Spectrom. Rev. 20, 157 (2001). K.J. Welham et al., Rapid Commun. Mass Spectrom. 12, 176 (1998). Z.P. Wang et al., Rapid Commun. Mass Spectrom. 12, 456 (1998). B.J. Amiri-Eliasi et. al, Anal. Chem. 73, 5228 (2001).

MALDI: some spectra The direct analysis of viruses, bacteria, fungi and spores was demonstrated* Positive and negative ion spectra of E. coli *C. Fenselau et al. Mass Spectrom. Rev. 20, 157 (2001). K.J. Welham et al., Rapid Commun. Mass Spectrom. 12, 176 (1998). Z.P. Wang et al., Rapid Commun. Mass Spectrom. 12, 456 (1998). B.J. Amiri-Eliasi et. al, Anal. Chem. 73, 5228 (2001).

MALDI: some spectra Depending on samples different protocols for sample preparation are available From A.E. Clark et al, Clin. Microb. Rev. 26, 547 (2013)

CONCLUSION MALDI-TOF is a very powerful technique for the analysis of biomolecules and large organic molecules The mail goals actually are - better understanding of the physical process; - acquisition of more and more spectra in order to improve databases for analyte identification

Thank you for the attention!