uv matrix-assisted laser desorption ionization: …...uv matrix-assisted laser desorption...

UV Matrix-Assisted Laser Desorption

Ionization: Principles, Instrumentation, and

Applications

1. Introduction

Matrix-assisted laser desorption ionization (MALDI)is, together with electrospray ionization (ESI) (seeChapter 7 (this volume): Electrospray Ionization:Principles and Instrumentation), one of the mostwidely employed ionization techniques in biologicalmass spectrometry (MS). MALDI was developed inthe second half of the 1980s (1,2), and its first pub-lished applications coincided with the first reports ofESI analyses of proteins. Owing to a low degree ofanalyte fragmentation, MALDI and ESI are oftenreferred to as ‘‘soft’’ ionization techniques, and assuch both paved the way for one half of the 2002Chemistry Nobel Prize—awarded for the develop-ment of soft desorption ionization methods for massspectrometric analyses of biological macromolecules.The origins of MALDI lie within the laser

desorption (see this chapter (this volume): Laser De-sorption: Principles and Instrumentation) and otherdesorption methods (see Chapter 8 (this volume):Secondary Ionization Mass Spectrometry and FastAtom Bombardment: Principles and Instrumentation;Ion and Atom Guns; Massive Particle Bombardment;Continuous-Flow Fast Atom Bombardment; PulsedFast Atom Bombardment; Desorption by Cf-252 Fis-sion Fragments and Desorption by Ion Beams fromAccelerators), which had as goal the development ofsensitive techniques for the intact desorption/ioniza-tion of labile analytes. There are several reasons whyMALDI-MS has become so popular. Among theseare high analytical sensitivity, the applicability to awide range of different compounds, easy-to-interpretmass spectra, simple sample preparation protocols,and a relatively high tolerance towards contaminantsand salt contents in the sample.The underlying idea of MALDI is the intact co-

desorption of analyte molecules with a molecular ex-cess of matrix molecules, which exert a crucial impacton the overall characteristics of the desorption/ion-ization process. Choosing the right analyte–matrixsystem is pivotal in MALDI and has been thefocus of many studies for developing better MALDIanalysis protocols (see this chapter: Matrix-AssistedLaser Desorption Ionization: Matrix Design, Choice,and Application and the following tutorials). How-ever, most of these were ‘‘trial and error’’ studiesand some of the most successful matrices (e.g., 2,5-dihydroxybenzoic acid, 2,5 DHB, and a-cyano-4-hydroxycinnamic acid, CHCA) were found empiricallyrather than by prediction.The MALDI process as a whole is highly

convoluted. Although desorption and ionization are

often discussed separately (see Sections 4 and 5), oneneeds to be reminded that many aspects of bothprocesses are coupled and occur at the same time.Moreover, the exact desorption/ionization pathwayscritically depend on the material properties of thecompounds and on the laser irradiation parameters.

The lasers most often used for MALDI-MS arepulsed ultraviolet (UV) lasers with wavelengths closeto the maximum UV absorption of the matrix (e.g.,337 nm, nitrogen gas laser; and 355 nm, frequency-tripled Nd:YAG solid state laser) and pulse durationsof 1–10 ns. Other lasers and laser wavelengths can beused for MALDI-MS but less frequently owing togenerally inferior MALDI and/or laser performanceat these wavelengths. Hence, UV-MALDI at theabove wavelengths is the predominant MALDImethod in commercially available MALDI-MS in-strumentation and in the main application areas ofbiological mass spectrometry (e.g., in proteomics: seeChapter 1 (Volume 2): Proteomies: An Overview).

There are, however, specific applications, in whicha particular laser–matrix combination can be supe-rior to the typically employed MALDI protocols andsetups. For instance, specific UV-MALDI matricesonly work at lower laser wavelengths such as thefrequency-quadrupled Nd:YAG laser wavelength of266 nm whereas infrared (IR)-MALDI with its widerrange of matrices is often more successful in the intactdetection of labile and large molecules (see this chap-ter: Infrared Matrix-Assisted Laser Desorption Ioni-zation) (3,4).

MALDI ion sources can now be found on a varietyof MS instruments including those typically used forESI: quadrupole time-of-flight (Q-TOF) and ion trapinstruments. Nonetheless, axial TOF instruments aremost commonly used for many reasons (e.g., highanalytical sensitivity and robustness). MALDI massspectrometers are further discussed in Section 3.

2. Basic Aspects of Performing MALDI-MS

2.1 Sample Preparation

One of the main reasons for the popularity ofMALDI-MS is its ease of analysis—its simple sam-ple preparation, high tolerance to contaminants, andfacile ion generation. The simplest and most com-monly used of all sample preparations is called the‘‘dried droplet’’ method (5,6). This method consistsof two steps, the spotting of the analyte and matrixsolution on the MALDI target and the subsequentevaporation of the volatile solvents. Given that anal-yte and matrix solutions are usually prepared sepa-rately, the mixing of both solutions can be eitherdone prior to the spotting or directly on the MALDItarget by applying typically 0.5–1 mL of each solutionin short succession. The drying (i.e., the evaporationof the volatile solvents) in a normal lab environmentis perfectly adequate although a mild stream of air is

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Desorption by Photons

often used to promote the drying process. Typicalparameters for the ‘‘dried droplet’’ method can befound in Table 1.The distinctive feature of MALDI, the matrix,

needs to provide sufficient energy absorption at thechosen laser wavelength. Besides this pivotal role,some further important functions must be providedby a functional matrix. Among these is the sufficient‘‘dilution’’ of analyte molecules in the matrix crystalto prevent analyte agglomeration and to facilitatemolecular desorption. For peptides, for example, theuseful analyte-to-matrix ratio range is roughly be-tween 5� 10ÿ2 and B10ÿ8. This means that even lessthan femtomole amounts of analyte can be sufficientfor successful analysis. From a practical point ofview, analyte and matrix molecules must be soluble inthe same solvent system. After crystallization, thesamples should have a sufficiently low vacuum pres-sure to avoid fast evaporation in the ion source.Although in most cases, matrices are used thatprovide a crystalline MALDI sample, other matrixsystems are available, in which the analyte com-pounds are not embedded in a matrix crystal butdesorption is rather initiated from a liquid matrix–analyte solution (see this chapter: Ionic Liquids asMatrices for Matrix-Assisted Laser Desorption Ioni-zation and Refs (7,8)) or from a pressed pellet prep-aration of finely ground analyte and matrix powder(see this chapter: Solvent-Free Matrix-Assisted LaserDesorption Ionization). Being by far the most widelyused preparation method, only crystalline analyte–matrix systems are further considered in this article.In some applications, ‘‘co-matrices’’ and additives

can improve the analytical performance (see thischapter: Matrix-Assisted Laser Desorption Ionization:Matrix Design, Choice, and Application). Most add-itives are employed either to enhance or to minimizeparticular types of ionization. For example, salts arefrequently applied to improve the detection of syn-thetic polymers in their cationized form, whereas

ammonium compounds are often employed to avoidcationization (e.g., of oligonucleotides) and promoteanalyte protonation (see this chapter: Matrix-Assisted Laser Desorption Ionization: Matrix Design,Choice, and Application).

2.2 Data Acquisition

MALDI-MS can be performed in either the positive-or the negative-ion mode, measuring positive ornegative ions, respectively. In most MALDI-MS ap-plications and instruments, however, the positive-ionmode usually provides higher sensitivity and spectralquality.

Once the MALDI sample is inserted into the massspectrometer and the chosen potentials (high vol-tages) have been applied, the main parameter thatneeds to be optimized either individually by the op-erator or via a computer-controlled fuzzy logic is thelaser intensity. In axial TOF instruments (see Section3.1), the laser fluence (i.e., pulse energy per irradiatedarea) critically determines the mass-resolving powersuch that it is typically adjusted to values only some-what above the ion detection ‘‘threshold.’’ For thecorresponding laser energy ‘‘threshold,’’ the value isusually determined by significant analyte ion detec-tion above the noise level (i.e., a ratio of analytesignal-to-noise of at least 2:1). The laser energy istypically adjusted to values slightly (10–30%) abovethis ‘‘threshold’’ value as a compromise betweenmaximizing the analyte signal while keeping the‘‘chemical’’ noise and matrix ion signals low (see be-low). This setting will allow the detection of a suf-ficient number of ions, generating ion signals withgood signal-to-noise ratio and ideally a symmetricalmonoisotopic ion profile. To improve the spectralquality further, up to a hundred or more spectra areusually accumulated. In axial TOF instruments theaccumulation of spectra, however, can also result in a

Table 1Typical irradiation parameters and sample preparation protocol for UV-MALDI

Laser wavelength: 337 nm (nitrogen laser)355 nm (frequency-tripled Nd:YAG laser)

Laser focus: 20–200 mm (diameter)Laser fluence: 50–500 Jmÿ2

Laser pulse duration: 1–10 nsLaser irradiance: 1� 106–5� 107Wcmÿ2

‘‘Dried droplet’’ sample preparation protocol:Mix 0.5–1mL of analyte solution with 0.5–1mL of 10 gLÿ1 matrix solution directly on the sample holder (target) anddry

Matrices (for polypeptide analysis):2,5-Dihydroxbenzoic acid (2,5-DHB)a-Cyano-4-hydroxycinnamic acid (CHCA)Matrix/analyte solvents:Water, acetonitrile and various solvent mixtures

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UV Matrix-Assisted Laser Desorption Ionization: Principles, Instrumentation, and Applications

decrease of the mass resolution and accuracy due tothe changing topological properties of the samplebetween laser shots.Whereas some MALDI preparation methods pro-

duce spatially rather homogeneous analyte distribu-tions, matrices like 2,5-DHB and 3-hydroxypicolinicacid (a matrix widely used in the analysis of oligo-nucleotides) come along with a typical ‘‘sweet spot’’effect. The ‘‘sweet spot’’ is a position at which a par-ticularly high analyte ion signal is obtained whileanalyte ion signals may be low or even absent at otherspots.Ionization efficiencies can substantially differ be-

tween compounds and, for example, depend on theamino acid composition in the case of peptides. Ana-lyte molecules with relatively low ionization proba-bility may therefore be suppressed in the presence ofmore easily ionized species. In the case of complexmixtures in particular, sample fractionation (inconcert with sample cleanup and concentration) bychromatography prior to MALDI analysis can greatlyenhance the ionization efficiency (see Chapter 3(Volume 8): Coupling of LC and MALDI-MS: Tech-niques, Instrumentation, and Application).Ion suppression also affects and complicates quan-

tification by MALDI-MS. Other factors makingquantification difficult are the variations in samplemorphology and composition and a possibly non-linear response of the signal intensity to a changinganalyte concentration. For these reasons, MALDI-MS is generally a much less suitable method forquantitative analysis than other analytical techniquesincluding ESI-MS (e.g., see Chapter 1 (Volume 2):Quantitative Proteomics via Stable Isotope Dilutionand Chapter 5 (Volume 3): Quantitative Analysis ofBiological Samples). However, the addition of areference analyte with similar ionization propertiesas the compounds under investigation or the use ofliquid matrices may facilitate quantitative analysis.

2.3 Data Analysis

Although specific aspects of data analysis aredescribed in more details in the respective MALDIapplication articles, it is important to discuss some ofthe basics here. In general, one needs to be awareof the instrumental and process-inherent effects thatoften concomitantly influence the final spectrum andits quality. Some of the main features that can oftenbe seen in MALDI mass spectra are shown in Fig. 1a.It is a typical positive-ion mode axial reflector TOFmass spectrum of a peptide mixture.One of the main characteristics of MALDI-MS is

the detection of predominately singly charged intactquasimolecular ions such as protonated molecules(e.g., [AþH]þ as seen in Fig. 1a,b,). Depending onthe analyte and the matrix and sample purity, othersingly charged quasi-molecular ions such as sodiatedand potassiated molecules ([AþNa]þ and [AþK]þ )

are also frequently seen. Particularly in the analysis ofglycans and synthetic polymers, metal-cationizedanalyte ions are preferentially formed and detected(see Fig. 1c), whereas cation adduct ions such as[AþNa]þ and [AþK]þ are undesirable in the ana-lysis of peptides and proteins, whose predominant ionformation channel is based on protonation.

Although singly charged analyte ions are predom-inately seen in MALDI-MS, multiply charged speciesare sometimes observed. Particularly, larger moleculesabove 10 000Da can accommodate more than justone charge without fragmenting during the analysis.This behavior is demonstrated in Fig. 1d showing themass spectrum of human transferrin, an approxi-mately 80 kDa protein.

Another important feature of MALDI is the gene-ration of matrix ions. At low to intermediate laserfluences, these ions are readily observable as distinctspecies in the low matrix mass range (Fig. 1a). Rad-ical matrix ions Mdþ, protonated matrix molecules[MþH]þ , and matrix ions undergoing a loss of wa-ter [M ÿ H2OþH]þ often form the base peaks in thepositive-ion mode, whereas either Mdÿ, [M ÿ H]ÿ, orMÿ 2H�dÿ matrix ions typically give rise to the basepeaks in the negative-ion mode. The exact composi-tion of this set of ions largely depends on the matrixused. Due to their high photochemical reactivity,many decomposition products are also formed. Withincreasing laser intensity, abundant matrix clusterions are generated that eventually produce a strongand often barely resolved background (Fig. 1b). Inaxial TOFMS, this unspecific background caneventually stretch over the entire low-to-intermediatemass range and can render the detection of genuineanalyte ion signal in the low mass range difficult.

Matrix oligomers and clusters that are initiallyformed upon material ejection also undergo fragmen-tation reactions. All types of fragments includingthose from matrix, analyte, and contaminant-relatedions contribute to the unspecific ‘‘chemical’’ back-ground or noise level. In many cases, this ‘‘chemical’’noise determines the limit of detection and the ana-lytical sensitivity. This is a particular problem in axialTOF instruments where the elimination of thesesources of ‘‘chemical’’ noise is limited.

2.4 Fragmentation

Although MALDI is a ‘‘soft’’ ionization technique,there is still scope for fragmentation. This can, forinstance, be initiated by high-energy collisions withplume constituents during the initial acceleration inthe ion source. The considerable amount of internalenergy imparted to the molecules during MALDI,however, inevitably leads to a certain degree ofmainly unimolecular fragmentation. This type offragmentation can occur at different times after thedesorption event and is often called metastable decaybecause most of this fragmentation is not prompt.

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Metastable decay significantly contributes to the‘‘chemical’’ background noise.Depending on the specific time point, metastable

decay occurs at different locations within the massspectrometer. If the decay occurs after the ions haveleft the source region, it is commonly called post-source decay (PSD) (see Chapter 3 (Volume 1): TOFand RTOF). PSD leads to unfocused (unresolved)lower molecular fragment ion signals in conventionalreflector TOF mass spectrometers (e.g., see, Fig. 1b).Hence, taking the increased ‘‘chemical’’ noise intoaccount, metastable decay can seriously restrict theanalytical performance. As a consequence, manymass spectrometer designs incorporate elements forthe elimination of metastable decay unless they areused for the acquisition of fragmentation spectra(e.g., tandem mass spectrometry—see Section 2.5).The type of analyte, choice of matrix, and the ion

extraction conditions also affect the degree of meta-stable decay. With respect to the matrix, one dis-tinguishes between ‘‘hot’’ and ‘‘cold’’ indicating thehigher and lower degree of ion fragmentation, re-spectively (see this chapter: Matrix-Assisted LaserDesorption Ionization: Matrix Design, Choice, andApplication). Metastable decay can range from a fewpercent, as typical for peptides that are analyzed byusing ‘‘cold’’ matrices, to values eventually approa-ching hundred percent (e.g., when using ‘‘hot’’ ma-trices in the case of sialylated glycopeptides).In axial TOFMS, ions spend most of their flight

time in the field-free drift region and, thus, pre-dominantly undergo unimolecular fragmentationthere (Fig. 2). Given that these fragment ions havethe same velocity as their precursors, and the overallflight time is identical for both species, evencompounds, which are very labile under MALDIconditions (e.g., oligonucleotides and sialylatedglycans), can still be analyzed in linear TOF mode(green ion trajectory in Fig. 2a). Metastable fragmentions may also be separated according to their m/zvalue in additional electrical fields, as in a reflector(red trajectory in Fig. 2b), and be used to obtainstructural information. This forms the basis of PSDanalysis (see Chapter 2 (Volume 2): Post Source De-cay MALDI in Peptide Analysis) and is further dis-cussed in Sections 2.5 and 3.1.Although it is common to classify fragmentation

according to its occurrence either by time or locationin the mass spectrometer, one needs to be remindedthat various types of fragmentation can be present.While it is evident that PSD mainly consists ofmetastable decay, others such as prompt fragmenta-tion and in-source decay (ISD) can lead to signi-ficantly different fragmentation spectra in axial TOFinstruments. Moreover, the types of dominantfragmentation in these cases are not necessarilyunimolecular.Prompt fragmentation (at the time of the desorp-

tion event) and ISD (shortly after the desorption

event; i.e.,o500 ns) (9,10) occur at much shorter timescales than PSD and well within the time frame of ionextraction. Therefore, the resulting fragment ions areusually well focused in all MALDI instruments,making it sometimes difficult to decide whether theassociated ion signals are due to fragmentation, pre-fragmentation that occurred during sample prepara-tion, or to genuine sample components. Obviously,all types and classes of MALDI fragmentation areimportant for the design of MALDI-MS and tandemMS (MS–MS) instruments.

2.5 Tandem Mass Spectrometry

For MALDI-MS–MS, both the naturally occurringdecay and operator-controlled dissociation are com-monly utilized. After MALDI was invented, PSD wassoon exploited as the first method for obtaining struc-tural information for peptides (e.g., see Chapter 2(Volume 2): Post-source Decay MALDI in PeptideAnalysis). To achieve increased fragmentation in PSDanalysis, ‘‘hot’’ matrices and high laser intensities wellabove the precursor ion detection ‘‘threshold’’ areusually used. For peptide sequencing, PSD of singlycharged MALDI ions predominately leads to complexensembles of a-, b-, and y-type fragment ions andmany internal fragment and immonium ions.

One alternative to PSD is ISD analysis, which isoccasionally used in axial TOF instruments (9,10).The main ISD fragment ions, particularly for largerpolypeptides, are c-, z-, and y-type ions. ISD product-ion spectra are remarkably similar to those obtainedby electron-capture dissociation (see Chapter 2 (Vol-ume 2): Top-down Approach to Characterization ofProteins) (11). An advantage of ISD is that largerpolypeptides and even proteins can also be analyzed,whereas the practical upper mass limit for PSD anal-ysis is about 3000Da. ISD spectra are usually lesscomplex than PSD spectra but the sensitivity is typ-ically far inferior.

There are other methods that utilize PSD by ap-plying analyte derivatization to direct the decay intospecific fragmentation channels. For instance, pep-tide charge-derivatization using sulfonic acid func-tionalities at the N-terminus takes advantage of a‘‘mobile’’ proton that promotes amide peptide back-bone cleavage and, thus, predominately afford y-typefragments (12). Fragmentation is not only increasedbut also exceedingly specific resulting in vastly in-creased sensitivity and simpler fragment ion spectrathat are easy to interpret (see Chapter 1 (Volume 2):Derivatization of Peptides for Analysis).

Lastly, commonly used and more generic dissocia-tion techniques can also be employed in MALDI-MS–MS. In particular, low-energy (kinetic particle energyof less than 500 eV) collision-induced dissociation(CID) can be employed (e.g., see Chapter 2 (Volume2): Sequence Analysis: Low Energy MS–MS–Peptide

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[A6+H]+

[A6+H]+

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+

[A8+H]+

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[A+3H]3+

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(c)

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[A+Na]+

Sequence Interpretation). In Q-TOF mass spectro-meters that have both an ESI and a MALDI ionsource, low-energy CID can be applied to ions formedby either ESI or MALDI (see Section 3.2). The typicaldifference in the charge-state of the ions formedby these two ionization techniques, however, results insignificantly different product-ion spectra (13). Singlycharged peptide ions, as are usually formed byMALDI, lead to a more complex product-ion spec-trum with fewer y-type ions than in spectra of multiplycharged peptide ions, which are common for ESI.MALDI axial TOF instruments offer the opportunityto perform high-energy CID owing to the high kineticenergy imparted to the ions in the ion source duringtheir extraction (see Fig. 2c). High-energy CID facil-itates charge-remote fragmentation (see Chapter 4(Volume 4): Charge-Remote Fragmentation: Applica-tion and Mechanism) and can provide structuralinformation that is not available with low-energyCID. For example, v-, w-, and d-type peptidefragment ions are virtually impossible to detect withlow-energy CID whereas high-energy CID can pro-mote these side-chain cleavages, enabling the distinc-tion between leucine and isoleucine and other isomers(see Chapter 2 (Volume 2): Sequence Analysis – HighEnergy MS–MS).

3. MALDI Instrumentation

The pulsed nature of MALDI and its historical con-nection to laser desorption have made axial TOF massspectrometers the natural choice for early MALDI-MS instruments. Later MALDI mass spectrometersutilized the great advances made in MS instrumenta-tion as a result of the importance of biological MS in

the bioanalytical sciences. Typical examples for thissecond generation of MALDI instruments are massspectrometers that minimize the impact of the initialion velocity on MS performance such as orthogonalTOF and ion-trap instruments.

3.1 Axial Time-of-Flight Mass Spectrometers

The transition from a laser desorption instrument toa MALDI instrument is seamless and requires nochanges to the instrumentation (see Fig. 2). To ac-commodate and exploit fully the analytical power ofMALDI, however, some changes are often made.

The use of a matrix usually leads to strong matrixion signals in the low molecular mass range (seeabove and Fig. 1a,b). Particularly in high sensitivitymeasurements with a high matrix-to-analyte ratioand increased laser intensity, these signals can lead todetector saturation and low detector response forsubsequent ions that strike the detector. Severalmethods have been devised either to remove abun-dant low molecular ions from the analyzer (e.g.,through ion deflection—see ion deflectors in Fig. 2a)which eliminates their detection, or to lower tempo-rarily the detector’s supply voltage and, thus, its re-sponse for this mass range. Both methods result inlow matrix ion signals but enhanced detector re-sponse in the analyte ion mass range. The lattermethod is typically applied in linear TOFMS becauseit also reduces the response to neutral particles poten-tially striking the detector.

The practical mass range for MALDI-generatedions when compared to laser desorption is far greater.This extended mass range also requires detector con-figurations enhanced for high mass detection. Many

Figure 1Axial MALDI-TOF mass spectra using a nitrogen gas laser with a wavelength of 337 nm (positive-ion mode). (a)Reflector mode mass spectrum of a peptide mixture with CHCA as matrix (A1, angiotensin II; A2, angiotensin I; A3,substance P; A4, bombesin; A5, renin substrate tetradecapeptide; A6, ACTH clip [1–17]; A7, ACTH clip [18–39]; A8,ITAM g-chain pY-peptide; A9, somastatin 28; M, matrix). The spectrum was acquired with a laser fluence slightlyabove the ion detection threshold (see text for details). (b) Reflector mode mass spectrum of a peptide mixture usingCHCA as matrix (A0, bradykinin; A1, angiotensin II; A2, angiotensin I; A3, substance P; A4, bombesin; A5, reninsubstrate tetradecapeptide; A6, ACTH clip [1–17]; A7, ACTH clip [18–39]; A8, ITAM g-chain pY-peptide; A9,somastatin 28). The spectrum was acquired with a laser fluence well above the ion detection threshold (see text fordetails). As a consequence, matrix ion signals are elevated and saturated, matrix ion clusters are formed and(metastable) analyte decay is increased as demonstrated by the loss of water for some peptides (e.g., substance P andACTH clip [1–17]), the significant fragmentation of the labile ITAM g-chain pY-peptide and the relatively lower ionsignals for high-mass peptides. (c) Reflector mode mass spectrum of PEG 3400 with 2,5-DHB as matrix. The additionof small amounts of KBr enhances PEG ionization and leads almost exclusively to potassium cationization. Forpeptides, similar amounts of KBr would, if at all, only slightly shift the ionization from protonation to potassiumcationization. The inset shows two specific polymers with the mass difference Dm of 44Da, corresponding to the massof the ethylene glycol repeating unit. Some sodium cationization is evident by ion signals 16Da lower than thepotassiated ion signals. Protonated molecule signals, however, are insignificant as indicated by the arrows forDm ¼ 38 Da. (d) Linear mode mass spectrum of human transferrin with 2,5-DHB as matrix. The spectrum clearlyshows the multiply charged transferrin ions ([Aþ 3H]3þ and [Aþ 2H]2þ ) with the singly charged monomer([AþH]þ ) still being most abundant whereas the singly charged dimer ([2AþH]þ ) is barely observable.

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t1 = tPSD t2 = tLIFT

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detectors based on electron cascade generation onlyprovide a low secondary ion/electron yield and,hence, a low ion signal when high-mass ions strikethe detector with low velocity. To increase the sec-ondary ion/electron yield, a ‘‘post-acceleration’’dynode is often mounted separately in front of thedetector. The application of a high potential to thisdynode gives the ions a more energetic impact and ahigher secondary ion/electron yield (i.e., higher ionsignal; see also Chapter 3 (Volume 1): TOF andRTOF). ‘‘Post-acceleration’’ is essential for the anal-ysis of large biomolecules above 50–100 kDa. How-ever, most commercial instruments are not equippedwith ‘‘post-acceleration,’’ owing to the decreasedmass resolution and accuracy, its greater technicaldemands, higher costs, and little benefit for the de-tection of low molecular ions such as peptide ions(e.g., as in proteomics).Given that axial TOF mass spectrometers are usu-

ally operated with high extraction fields in the ionsource, the entire instrument needs to be under highvacuum (typically o10ÿ6hPa). High extraction fieldstrengths and potentials provide advantages for iondetection (see above) and overall instrumentalperformance, particularly with respect to time-lag fo-cusing using delayed ion extraction. The introductionof delayed ion extraction using pulsed high voltagesin the mid-1990s tremendously increased both themass resolving power (410 000) and the mass accu-racy (10–50 ppm) (see Chapter 3 (Volume 1): TOFand RTOF).MS–MS on axial TOF mass spectrometers is usu-

ally undertaken by using PSD or CID (Fig. 2b,c). Inboth cases, a precursor ion selector is employed toselect ions with a given TOF acquired in the first driftregion. These ions are subjected to CID in a collisionchamber filled with background gas (e.g., argon)whereas for PSD, no collision gas is required. Inconventional reflector mass spectrometers (e.g., thoseequipped with a double-stage or gridless reflector),

however, PSD ions are not satisfactorily focused,let alone being reflected on the detector in the case oflow mass fragment ions. Hence, the reflector voltageneeds to be stepped through several focal ranges, andspectra acquired in each range need to be stitchedtogether to obtain a sufficiently resolved PSD prod-uct-ion spectrum (see Chapter 3 (Volume 1): TOF andRTOF and Chapter 2 (Volume 2): Postsource DecayMALDI in Peptide Analysis). To avoid this laboriousand sample- consuming acquisition, alternativereflector TOF designs can be employed. In quadraticand curved field reflectors, most PSD fragment ionsare focused, and no spectra need to be stitchedtogether (see Chapter 3 (Volume 1): TOF and RTOF).The angle of acceptance for these reflectors, however,is far lower than for conventional reflectors, makingthem less sensitive. Moreover, PSD fragment ions arealso focused and seen in the normal mass spectrum,which is undesirable for analyses of complex analytemixtures. An alternative to higher order reflectordesigns is based on increasing the kinetic energy ofthe PSD fragment ions in a potential lift cell beforethey enter the reflector (see Fig. 2b) (14). If, after thepotential lift, all PSD fragment ions possess at least60–70% of the kinetic energy of their precursor ion,even conventional reflectors are able to adequatelyreflect and focus them on the detector. A majordisadvantage of the PSD method is that mass accu-racy is relatively low (B0.2Da) even under optimalconditions.

3.2 Orthogonal Time-of-Flight Mass Spectrometers

With the rise of proteomics, new developments in MSinstrumentation and their integration in complexbioanalytical workflows became necessary. As a con-sequence, new types of MALDI mass spectrometerswith a general emphasis on the hybridization andhyphenation of technologies were developed. In 2000,

Figure 2Schemes of MALDI-TOF mass spectrometers. (a) Axial MALDI reflector TOF mass spectrometer. After ionextraction ions are further guided and focused before entering the field-free drift region. Matrix ion signals can beminimized by using ion deflection plates and/or by temporarily lowering the detector voltage to avoid detectorsaturation. In the reflector mode, energy-focusing increases the mass resolving power, which can be further enhancedby time-lag focusing using delayed ion extraction (see Chapter 3 (Volume 1): TOF and RTOF). (b) Axial MALDI-PSDTOF-TOF tandem mass spectrometer. Ions may fragment through metastable decay in the first drift region (t1). Afterion selection, the kinetic energy of the fragment ions (and unfragmented precursor ions) will be increased through apotential lift (t2) sufficiently strong for all ions to be reflected and focused on the reflector detector (see text for details).Alternatively, a set of spectra recorded at stepwise-lowered reflector voltages can be stitched together. In this case andfor higher order reflector designs that are capable of focusing all PSD fragment ions, no potential lift cell is required.(c) Axial MALDI-CID TOF-TOF tandem mass spectrometer. From the ion selector, transmitted precursor ions arefragmented by collisional activation in the collision cell (t2). The fragment ions are then conventionally separated andanalyzed by the subsequent reflector TOF mass analyzer. (d) Orthogonal MALDI-(CID) Q-TOF (tandem) massspectrometer. After MALDI ions are formed, collisional cooling thermalizes their initial ion velocity. Ions areorthogonally injected into the TOF ion pusher region and conventionally analyzed by TOFMS at high repetitionfrequencies. Precursor ion selection and fragmentation in a CID cell facilitate MS–MS measurements.

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several publications reported the coupling of MALDIion sources to orthogonal TOF and hybrid Q-TOFinstruments (Fig. 2d) (15–17). These instruments em-ploy effective collisional cooling and ion manipula-tion to dampen the ion motions in all directions,reducing the initial ion velocity distribution and in-ternal ion energy acquired from the MALDI processand, consequently, enhancing ion survival rates andimproving the ion beam quality and overall MS per-formance (18). When the ions enter the TOF massanalyzer after orthogonal injection (see Fig. 2d), theinitial velocity distribution is marginal and MS per-formance is largely dependent on the analyzer func-tion and less on the ionization process or ion sourceconditions. Mass spectral parameters such as massresolving power, mass measurement accuracy andprecision are virtually the same whether MALDI orESI ions are analyzed.The relatively high ion source pressure of typically

more than 10ÿ2 hPa used for collisional cooling alsofacilitates the use of the same mass spectrometer forboth ESI and MALDI with an easy swapping of ionsources. Even the use of atmospheric pressure (AP)-MALDI ion sources is possible (15) (see this chapter:Atmospheric Pressure Matrix Assisted Laser Desorp-tion Ionization (AP-MALDI)).Other advantages of effective collisional cooling

and decoupling of the ion source from the TOFanalyzer include: (i) the generation of a quasicontin-uous ion beam enabling ESI beam-like measurementswith high repetition TOF analysis and low peak sat-uration; (ii) low metastable decay, particularly at thetime of the TOF measurement, producing only fewadditional ion signals and low signal backgrounddue to ‘‘chemical’’ noise; and (iii) a low influence ofMALDI conditions on the TOF measurement, offer-ing a greater choice of sample preparations and des-orption parameter values.Nonetheless, sensitivity is limited by the duty

cycle of the pulsed TOF measurements from the qua-sicontinuous ion beam and, thus, theoretically lowerthan in axial TOF instruments, although improve-ments can be achieved by ion trapping and ioninjection coordinated with the TOF pulsing (19). Inaddition, the low ion acceleration voltages in mostorthogonal TOF instruments discriminate the detec-tion of the larger singly charged MALDI ions.Another potential disadvantage of collisional cool-ing is the formation of adduct complexes betweenanalyte ions and matrix molecules. The extent of suchreactions can be considerably higher than in high-vacuum MALDI ion sources as employed in axialTOF mass spectrometers.One important practical point that also needs to be

considered for the employment of MALDI sourceson instruments that were originally designed for ESI-MS and in which quadrupoles (or multipoles) areused, is the predominantly single charge state ofMALDI-generated ions. In contrast to the generally

multiply charged ESI ions that can mostly be analy-zed (or transmitted) by quadrupoles (or multipoles)with small m/z ranges (typically m/z 0–4000) MALDIions require quadrupoles with extended m/z ranges.Given that there are practical limits to the m/z rangeof a quadrupole in both sensitivity and duty cycle,most large-range quadrupoles are limited to an m/zrange of up to 16 000 being also the overall upperlimit for the instrument and, thus, by far lower thanthat of axial TOF mass analyzers. This is of partic-ular importance for MS–MS instruments, in whichusually quadrupoles (or multipoles) are employed forprecursor ion selection.

Nonetheless, one of the advantages of hybridorthogonal TOF instruments (e.g., Q-TOF) is theirsuperior performance in MS–MS. The TOF perform-ance is virtually unchanged between MS and MS–MSmeasurements, providing high mass resolvingpower and accuracy even in the MS–MS mode. Aswith ESI, CID-MS–MS measurements of MALDI-generated precursor ions can be successfully per-formed at high sensitivity (low femtomole amountsof sample) with efficient precursor ion selection(unit mass resolution) and low ‘‘chemical’’ noisebackground.

3.3 Ion Trap Mass Spectrometers

MALDI in combination with Paul ion trap mass spec-trometers, also called quadrupole ion trap mass spec-trometers, was reported in the early 1990s (20–22) (fordetails on Paul ion traps, see Chapter 3 (Volume 1):Ion Traps). As with two-dimensional quadrupoles, thewide initial velocity distribution of MALDI ionsreduces the analytical performance (i.e., the trappingefficiency). Therefore, efficient dampening of the ionmotions is also vital for MALDI with Paul ion traps.Abundant matrix ions pose another problem and needto be excluded from the trapping because ion trapshave a limit for the maximum number of ions theycan hold before space-charge effects become signifi-cant. In addition, metastable decay can be detrimentalto the MS analysis considering the relatively longanalysis time in ion traps.

Nonetheless, ion traps are compatible with MALDIif the above challenges are satisfactorily met. To ac-hieve this goal, an external ion source with adequateion transfer lines and collisional cooling is typicallyemployed whereas the pulsed nature of ion trappingcan be synchronized with the pulsed formation ofMALDI ions and their transfer to the ion trap.

Similarly to quadrupole and Q-TOF mass anal-yzers, however, ion traps have limitations for theanalysis of high m/z ions. Today’s upper limit forPaul ion traps is approximately m/z 4000–6000. Inprinciple, MALDI ions above an m/z value of 10 000can also be detected on Paul ion traps, but only atreduced performance.

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Another ion trap mass spectrometer is the Fouriertransform ion cyclotron resonance (FTICR) instru-ment (see Chapter 3 (Volume 1): Fourier TransformIon Cyclotron Resonance). Successful coupling ofMALDI to FTICR instruments was first achieved inthe early 1990s. In general, for MALDI-FTICR-MSone has to consider the same issues as for MALDI-MS employing Paul ion traps. The required ultrahighvacuum (around 10ÿ10 hPa) and even longer analysistimes than in Paul ion traps pose additional demandson the coupling. Owing to the high kinetic energyof MALDI ions and their metastable ion decay,FTICR-MS performance was initially poor. Withadequate collisional cooling, external ion accumula-tion and/or ion deceleration, analyte ions can be suf-ficiently cooled and trapped while matrix ioninterference is minimized. At modest m/z value, theseadvances enable those advantages of FTICR as seenwith ESI (i.e., extremely high mass resolving powerand mass measurement accuracy at high sensitivity).The analytical performance, however, significantlydegrades with increasing m/z values, making ESI withits higher charge state ions the preferred ionizationtechnique for most applications. On the other hand,MALDI sources on FTICR mass spectrometers offerthe advantage of coordinating the formation ofMALDI ions with the FTICR analysis, avoidingtiming problems and superfluous sample consump-tion as it can be the case for ESI. Specifically, offlineliquid chromatography (LC)–MALDI coupled toFTICR ensures the detection of all LC-eluted com-ponents while online LC–ESI is prone to missingcomponents that elute while the FTICR instrumentis busy analyzing earlier eluents—a typical dead timeeffect. These issues become increasingly important inproteomic analyses, in which high proteome coverageis often required (see Volume 2).

4. The Desorption Process in UV-MALDI

MALDI is a complex process that involves opticaland mechanical phenomena as well as thermo-dynamic and physicochemical processes of phasetransition and ionization (23,24). Figure 3 depicts theMALDI process schematically. For conventionalMALDI employing crystalline matrices and typicallaser conditions, the desorption process is essentiallythermally controlled. However, as outlined below, theprocess takes place in thermal nonequilibrium.

4.1 Importance of Irradiation and MaterialParameters

To achieve optimal results, several material and irra-diation parameters (see Table 1) need to be adjustedcarefully. The laser-pulse duration, for example,must be sufficiently short to avoid thermal degrada-tion of the analyte compounds. In other words, time

constants for the desorption channel need to be smallcompared to those which induce thermal fragmen-tation. Typically, laser pulses in the low nanosecondrange are employed for UV-MALDI. Lasers witheven shorter pulses may be used, but generally do notlead to improved results.

The second most important irradiation parameter,the laser emission wavelength, must correspond toa high optical absorption of the matrix to achieve ahigh energy density in the sample. Suitable matri-ces exhibit molar absorptivities between 2000 and20 000Lmolÿ1 cmÿ1 at the UV excitation wave-lengths, as measured in solution. Assuming similarabsolute values for matrix crystals, the laser light willbe essentially absorbed within the upper 20–200 nmof the crystals. Typical matrices with conjugatedaromatic systems exhibit relatively broad absorptionbands of several tens of nm in width, easing thedemands on the exact choice of the laser. As notedabove and in the following article: Matrix Design,Choice, and Application, matrices with absorptionbands peaking in between B300 and 350 nm arealmost exclusively employed for UV-MALDI, match-ing well the emission wavelengths of both thenitrogen (337 nm) and/or the frequency-tripledNd:YAG laser (355 nm). Essentially all of the com-mon matrices like 2,5-DHB and cinnamic acidderivatives (e.g., CHCA and sinapinic acid) belongto this group. A noteworthy drawback of thefrequency-tripled Nd:YAG laser is a reduced opti-cal absorption for a few important matrices like3-hydroxypicolinic acid.

Low ‘‘sublimation’’ temperatures at which signifi-cant desorption sets in are often regarded as anadvantageous parameter that helps to reduce un-wanted thermal analyte degradation. This is one rea-son why no suitable matrix compound with extendedconjugate electronic system has been found so far. Themore extended systems shift the optical absorption

Neutral matrixmolecule

Neutral analyte molecule

Matrix ion

Analyte ion

Analyzer

Laser

Figure 3Scheme of the MALDI process.

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into the red (i.e., the visible region) but also increasethe intermolecular binding strength of the molecules.Even a high temporary lattice temperature of the ma-trix crystal will not automatically lead to a strong in-ternal excitation of analyte molecules. The frequencymismatch between lattice and intramolecular analytevibrations forms a bottleneck (25) that effectively de-lays a full equilibration. Evaporative and collisionalcooling within the expanding MALD(I) plume furtherstabilize labile analyte molecules.With a few exceptions such as molecules carrying

chromophores (e.g., cytochrome c), UV absorptionabove 300 nm is negligible for biomolecules and di-rect photodissociation of analyte molecules isavoided. For the following discussion, it can be as-sumed that, due to the low molar analyte-to-matrixratio, most physicochemical properties of the analyte-matrix crystals are essentially identical to those ofneat matrix preparations.

4.2 Energy Absorption and Relaxation

Population of the first excited singlet state (S1) byresonant optical absorption serves as the primary ex-citation step. Due to the Frank–Condon principle,some excess photon energy is at this stage alreadyconverted into heat by internal conversion to vibra-tionally lower lying S1 states. Fluorescence, inter-system crossing, ionization after absorption of asecond photon, and photochemical reactions lead tosome energy ‘‘loss,’’ but most of the energy stored inthe S1 state is transformed radiationless by internalconversion into intramolecular lattice vibrations (i.e.,heat). S1 decay constants can be estimated from fluo-rescence studies to be on the order of a few 10ÿ10 s(26). This is somewhat shorter than typical laser pulses.Thermal relaxation times in the condensed phase

tth can be estimated by

tth ¼rcp

althd2

where r is the density of the material, cp the specificheat, lth the thermal conductivity, and d is the laserpenetration depth. The factor a accounts for the size/geometry of the irradiated crystals and is usually be-tween 1 and 4. Using typical material parameters,values for tth on the order of B10 ns are calculated.This is close to, but still slightly above, the laser-pulseduration. Once all energy is absorbed, the excitedvolume will exhibit a relatively homogeneous tem-perature profile with sample depth while heat dissi-pation out of the excited volume is low.The laser heating is accompanied by rapid expan-

sion of the material and, hence, by the generation ofphotomechanical stress. Time frames for acousticenergy relaxation are determined to the first order bythe propagation time of a stress wave through theexcited medium (i.e., by the ratio of the excitation

depth d and the speed of sound vs: tac ¼ d=vs). Valuesfor tac are on the order of a few tens of picosecondsfor typical UV-MALDI conditions, and the stressamplitudes are, therefore, too low to have any majoreffect. Photoacoustic stress can, however, play animportant role in IR-MALDI where considerablylarger laser penetration depths are found (see thischapter: Infrared Matrix-Assisted Laser DesorptionIonization).

Laser fluences at the ion detection threshold are onthe order of a few tens to a few hundreds of joules persquare meter (Jmÿ2), depending on matrix absorp-tion and laser spot size (27). For typical laser spotsizes of 100–200 mm in diameter, this translates torather equal energy densities per volume for differentmatrices of approximately 1� 109 Jmÿ3 at the sur-face of the sample. These values are close to the sub-limation enthalpies. Energetically, vaporization ofthe surface volumes is therefore possible. Under equi-librium conditions, an energy per volume of1� 109 Jmÿ3 corresponds to a temperature increaseof some hundred Kelvin. The nonequilibrium char-acter of the process can lead to even higher latticetemperatures. Post-ionization studies show that atfluences below the ion detection threshold, a highnumber of neutral molecules are already ejected (27).

In the rarely used suspension matrices, where thematrix usually consists of a suspending compoundsuch as glycerol with suspended nanoparticles like co-balt or TiN, laser light absorption is decisively different(2) (see this chapter: Matrix-Assisted Laser DesorptionIonization: Matrix Design, Choice, and Application).Whereas the optical and thermodynamic properties ofmicrometer-sized particles do not differ much fromthose of the corresponding bulk material, nanoparti-cles with diameters below B100 nm can show differentfeatures due to surface effects like the excitation ofsurface plasmons. As a consequence, the opticalabsorption can become very high, even if the bulkmaterial only absorbs moderately. By using nanopar-ticle suspension matrices, temperatures on the order of10 000K are reached with high heating rates (28).

4.3 Material Ejection

At low laser fluences below or close to the ion de-tection threshold, layer-by-layer sublimation fromthe surface forms the dominant desorption mecha-nism (23). Desorption sets in during the laser pulseand, because of the evaporative cooling, rapidly ter-minates after the laser pulse has ceased. Moleculardesorption dominates this fluence regime, although a‘‘selvedge region’’ consisting of molecules and smallclusters is formed (29). Hence, a certain percentage ofclusters will also persist in the expanding plume.

The molecular desorption rate can be approxi-mated by an Arrhenius-like relation. Taking into ac-count that, for nanosecond pulsed lasers, the overall

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amount of ejected material can be set proportional tothe desorption rate at the peak temperature, theoverall desorption yield of monomeric molecules canbe described by a ‘‘quasithermal’’ equation of theform

YBPðAÞexpÿEa

kBðT0 þ BFÞ

� �

where Y is the ion yield, Ea the activation energy forthe process, T0 the initial sample temperature, and kBthe Boltzmann constant (27). F denotes the laser flu-ence, and B a conversion factor accounting for thetransformation of laser pulse energy into (lattice)temperature of the sample. P(A) is a pre-exponentialfactor that includes a (less strong) additional tem-perature dependence. For MALDI, there is a strongdependence of the ion yield on the laser spot size (27),which is also included in the prefactor.At higher laser fluences, where the desorption rate

is not sufficient to balance the energy deposition, siz-able overheating occurs, and the heated material canablate instantaneously by phase explosion (30). Inthis case, molecular desorption is accompanied by theejection of a larger number of clusters (29). Thismechanism can play a particularly significant role inhigh-pressure MALDI ion sources (see this chapter:Atmospheric Pressure Matrix Assisted Laser Desorp-tion Ionization (AP-MALDI)) for which high laserfluences are frequently used. In axial TOF instru-ments, in which a background pressure of approxi-mately 10ÿ7 hPa is normally maintained, the usefulMALDI fluence range is limited and typically reachesfrom the ion detection threshold to values of a factorof 2–3 above. Further increase of the laser fluenceresults in significant analyte fragmentation anddegradation of the quality of the mass spectra. Thiseffect is also believed to account in part for the strongspot size effect and over-proportional increase of thethreshold fluence with decreasing spot size.Whereas in conventional MALDI analyses, laser

focus diameters between 50 and 200mm are typicallyused, smaller diameters are important for imagingMALDI-MS applications, in which a high lateralresolution is sought (see this chapter: Surface Ana-lysis of Biomaterials and Chapter 4 (Volume 2): Pro-filing and Imaging Peptides and Proteins fromMammalian Tissue Sections by MALDI and Single-Cell Peptide Analysis with MALDI-MS). Althoughspot sizes with a few micrometers in diameter aretechnically easy to realize, they lead to conditionsthat are apparently too destructive for the analysis.Spot sizes approaching a few micrometers in dia-meter, the accompanied strong increase in thresholdfluence also leads to the ablation of a volume of ma-terial of several hundreds of nm in depth, thuschanging the overall processes considerably. MALDIimaging is therefore mostly performed with minimallaser spot sizes of about 20 mm in diameter.

4.4 MALDI Plume Expansion

In a typical setup with a focal laser spot size of100mm in diameter, a layer of material of about a fewto a few tens of nanometers in depth (i.e., a few to afew tens of monolayers) is desorbed per laser pulse atthe ion detection threshold. The ejected moleculesundergo a large number of intermolecular collisionsduring the phase disintegration and the plume ex-pansion. These collisions have a significant effect onthe expansion characteristics and lead to a forward-shaped MALDI plume. Average axial velocities (i.e.,in the direction of ion extraction) of neutral matrixmolecules are on the order of 500m sÿ1 with a widthof the velocity distribution being of a similar size (23).Mean velocities of MALDI ions are even higher andapproach 1000m sÿ1 (31,32). Thus, the ions are es-sentially found in the front part of the expandingMALDI plume. Radial velocity components reflectthermal values more closely and, thus, are lower thanthe axial ones and more mass dependent (32). Mul-tiple collisions with matrix molecules accelerate theentrained analyte molecules to similar axial velocities(31). For large analyte molecules, this results in highkinetic energies on the order of several eletronvolts,again with similar widths of the energy distribution.This leads to a sizable reduction of the performanceof axial TOF instruments and problems in ion trans-mission and ion trapping in quadrupole and ion trapmass spectrometers, respectively.

For high-pressure ion sources (see Sections 3.2 and3.3), which are typically operated at a pressure ofabout 1 hPa and up to 1000 hPa for atmosphericpressure (AP)-MALDI (see this chapter: AtmosphericPressure Matrix Assisted Laser Desorption Ionization(AP-MALDI)), collisions with the background gaslead to a rapid thermalization and a very differentplume expansion character. The accompanied colli-sional cooling leads to a stabilization of co-desorbedanalyte molecules and allows the effective use of Q-TOF and ion trap instruments for MALDI-MS (seeSections 3.2 and 3.3) even at very high laser fluences.

5. The Ionization Process in UV-MALDI

One feature that makes MALDI so attractive is thatthe method works well for an extraordinarily widerange of chemical compounds possessing rather dis-similar physicochemical properties. For example,MALDI can be applied to virtually all classes of bio-molecules (see Volumes 2 and 3), but also to syntheticpolymers (see Chapter 11 (this volume): SyntheticPolymers), and to many inorganics. The exact ioniza-tion pathway depends on the physicochemical pro-perties of analyte and matrix, in particular protonaffinities (PAs), gas-phase acidities (GPAs), and gas-phase basicities (GPBs). Given that these vary withthe type of analyte, the choice of the matrix should be

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properly adjusted to the type of compounds underinvestigation (see this chapter: Matrix-Assisted LaserDesorption Ionization: Matrix Design, Choice, andApplication). The convoluted processes of desorptionand ionization, involving the rapid phase transition,temporary high temperatures, and manifold energeticcollisions, render it difficult to establish a simple pic-ture of the MALDI ionization. In many cases, thematrix plays a vital role in the ionization process (e.g.,as proton donor or acceptor). Owing to the highnumber of intermolecular collisions during the plumeexpansion, energetically controlled thermochemistrywill in general determine the mass spectrum. Reac-tions thermochemically not feasible under equilibriumconditions may contribute to the overall outcome. Asa consequence, surprisingly similar mass spectra (inthe analyte mass region) are produced by UV- and IR-MALDI despite the very different primary ionizationmechanisms in the two wavelength ranges (see thischapter: Infrared Matrix-Assisted Laser DesorptionIonization). Only at very low laser fluences, at whichonly few ions are produced, the process will be kinet-ically rather than energetically controlled. The tran-sition from one regime to the other is in some casesreflected in a changing composition of matrix ionspecies (e.g., the ratio between radical ions and pro-tonated molecules) in the mass spectrum.The ion detection laser fluence threshold, at which a

sufficient number of ions is generated to produce amass spectrum, is higher by a factor of 2–3 than thatfor the detection of neutral molecules (27). Further,the dependence of the gas-phase yield on the laserfluence is stronger for ions than for neutral molecules,indicating an additional ionization process. Providedthere is sufficient analyte-to-matrix ratio and detec-tion sensitivity, analyte and matrix ions exhibit thesame fluence thresholds and the same yield depend-ence on laser fluence (27). Estimations for matrix ionyields (i.e., the ratio of generated matrix ions to over-all desorbed matrix molecules) range between 1:103

and 1:105. In cases of favorable secondary ionizationconditions, the analyte ion yield is likely to be on thehigh side of this range or even somewhat above.Analytes with molecular weights exceeding 10 kDa

are typically observed with sizeable yields of multiplycharged ions (protonated/deprotonated molecules)(see Fig. 1d). As molecules become larger in size, thecharges are sufficiently separated so that the Cou-lomb energy of the system becomes low, and thecharge sites more independent. The maximum possi-ble charge state scales with the size of the analyte.Very large molecules such as monoclonal antibodiesof molecular weight B150 kDa may carry as much asfive charges with sizable intensity. The high numberof collisions in the expanding MALDI plume alsohelps to overcome the Coulomb barrier in the pri-mary charge separation process.Although not fully separable, it is nevertheless

helpful to differentiate between primary excitation/

ionization of matrix molecules and reactions betweenthese species, on the one side, and secondary ioniza-tion reactions between them and the analytes, on theother. One should keep in mind that in addition tothese reaction schemes, involving essentially mono-meric molecular species, a high percentage of mole-cules, at least temporarily, form dimers, oligomers, oreven small clusters. Moreover, the initial matrix-analyte solid state provides an environment ratherdifferent from the final gas-phase state. This cansignificantly change the energetics (33,34).

5.1 Primary Ionization Reactions

Some possible primary ionization pathways are de-picted in Table 2a. Typical ionization energies (IEs)(see Chapter 1 (this volume): Ionization Energies) ofmonomeric matrix molecules are approximately 8 eV.Numerically, two photons of either 3.68 or 3.49 eV at337 and 355 nm, respectively, are not sufficient tocause ionization. Even though IEs can be lower by afew tens of electronvolt in the condensed phase owingto solvation effects, other processes must be involvedto balance the energy requirements. For example, thethermal energy content can assist the ionization. Thesecond excited singlet state (S2), from which ioniza-tion can most easily be initiated, can be populated bysubsequent resonant two-photon absorption. Thisprocess (reaction 2.1 in Table 2) however, probablydoes not apply at typical MALDI fluences of about100 Jmÿ2 (see Table 1).

Energy pooling between neighboring excited mole-cules or between two excitons (mobile electron–holepairs in the solid; reaction 2.2) forms the most likelyionization pathway. In a process called singlet–singlet(S1–S1) annihilation (26), one molecule is excited intothe second excited S2 state while the S1 state of thesecond molecule is relaxed. Energy pooling may alsotake place as a multicenter process involving morethan two molecules; this also loosens the require-ments on the energetics.

Whereas the lifetimes of the first excited singletstates in the solid are only approximately 10ÿ10 s fortypical matrix compounds, they can extend to a fewnanoseconds or more for monomeric molecules.Therefore, ionization that is delayed with respect tothe laser pulse can occur during the plume expansionphase. Intersystem crossing from a singlet to a long-lived triplet state may also extend the time windowfor ionization.

Excited state proton transfer (ESPT; reactions 2.3)and even ground state proton transfer reactions (dis-proportionation reactions; reactions 2.4) from neu-tral matrix molecules are also theoretically possibleprimary MALDI ionization mechanisms (35). Or-ganic molecules such as aromatic amines and phenolsfrequently exhibit an increased acidity upon elec-tronic excitation; the increased acidity could allowESPT to analyte. A sufficient yield of ground state

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Table 2Possible primary (a) and secondary (b) ion formation mechanisms in MALDI

Mechanism Reaction Comment

(a) Primary ionization mechanismsSingle moleculemultiphotonionization

Mþ ndhv-Mdþ þ eÿ ð2:1ÞFeasible but with low rates attypical laser fluence values

Energy pooling (S1–S1-annihilation) Mþ hv-M�

M� þM�-M�� þM

M��-Mdþ þ eÿ ð2:2Þ

Probably the major pathway forprimary matrix ionization

Excited-state protontransfer Mþ hv-M�

Feasible for matrices with a highacidity in the exicited state, butunlikely to be a general majorMALDI pathway

M� þM- ½MþH�þ þ ½MÿH�ÿ ð2:3aÞ

M� þA- ½AþH�þ þ ½MÿH�ÿ ð2:3bÞ

Ground-state protontransfer MþM- ½MþH�þ þ ½MÿH�ÿ ð2:4aÞ

MþA- ½AþH�þ þ ½MÿH�ÿ ð2:4bÞ

MþA- ½AÿH�ÿ þ ½MÿH�þ ð2:4cÞ

Sufficient proton transfer rateswould require unlikely highplume temperatures. Protontransfer from matrix oligomerswould energetically besomewhat less costly (33)

Thermal ionizationMþM-Mdþ þMdÿ ð2:5aÞ

M-Mdþ þ eÿ ð2:5bÞ

A relevant pathway fornanoparticle suspensionmatrices but not for commonMALDI

(b) Secondary ionization mechanismsProton transfer

½MþH�þ þA-Mþ ½AþH�þ ð2:6aÞ

½MÿH�ÿ þA-Mþ ½AÿH�ÿ ð2:6bÞ

Mdþ þA- ½MÿH�d þ ½AþH�þ ð2:6cÞ

The dominant ionizationpathway for major classes ofcompounds (e.g., peptides andproteins). Proton transfer mayproceed from a matrix ion in aground or excited state

Cationization otherthan protonation Aþ Catþ- ½Aþ Cat�þ ð2:7aÞ

Relevant where proton transfer isnot feasible

Aþ ½Mþ Cat�þ- ½Aþ Cat�þ þM ð2:7bÞ

Electron transferMdþ þA-MþAdþ ð2:8Þ

Except for special cases, not arelevant pathway

M, matrix molecule; M*, M**, singly and doubly excited matrix molecules; A, analyte molecule; Catþ metal cation.Note: Reactions indicated for matrix monomers may similarly also be initiated from a matrix dimer or oligomer.

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proton transfer reactions between two initially neu-tral matrix molecules, however, would require tem-peratures of a few thousand Kelvin. These transferswould be at best a small fraction provided by thehigh-side tail of the Boltzmann distributions. A sec-ond major argument against the general involvementof ESPT, is that some good matrices are clearlyESPT-inactive in solution and that many moleculeswith a high ESPT-activity fail completely as MALDImatrix. Proton transfer between a neutral matrix andan analyte molecule may also take place in a directreaction (2.4b and c), but the requirements on theenergetics are high, as discussed above. Although thelatter mechanisms cannot be completely excluded tocontribute to the overall ion yield, they are thereforenot very likely. Furthermore, many experimentalobservations support energy pooling reactions as themajor primary ionization mechanisms. This reaction(2.2) also explains the often detected radical ions ofthe matrix molecules.In a different scenario, ions that were preformed

already in the condensed phase are simply releasedupon desorption. With the exception of ionic com-pounds or metal-complexed analytes, however, thesolvent state is generally not reflected in the MALDImass spectra. Preformed negative ions are also rarelyobserved. Hence, for the majority of analyte com-pounds the ejection of preformed ions is either of littlerelevance, or initially desorbed ions are rapidly maskedby secondary reactions taking place in the plume.Thermal ionization (reaction 2.5) plays a decisive

role in laser desorption/ionization with nanoparticlesuspension matrices, where the temperature can reachpeak values of 10 000K or more. Ionization is typ-ically assisted by the suspending matrix compoundsuch as glycerol.

5.2 Secondary Ionization Reactions

Some possible secondary ion reactions are depicted inTable 2b. Energetically, proton transfer from either aradical matrix molecule, a protonated matrix molecule,or eventually a protonated matrix-oligomer to a neu-tral analyte molecule (reactions 2.6) are in many caseshighly favored (e.g., in the MALDI analysis of peptidesand proteins; see Fig. 1a,b,d) (24,36). In the negative-ion mode, the differences of the GPBs of deprotonatedanalytes and those of matrix anions determine whetherproton detachment from a neutral analyte molecule isenergetically feasible. Generally, these differences aresmaller for a particular analyte species and the matrixthan the differences in the PAs (see above) and theyield of [M ÿ H]ÿ ions, therefore, depends strongly onthe actual differences. For many matrices like 2,5-DHB, the yields of deprotonated analytes such aspeptides are typically substantially lower than thosefor their positively charged protonated counterpartsand—for the above reasons—depend more strongly onthe molecular composition of the analyte.

The PAs for some analyte molecules, for exampleoligosaccharides and synthetic polymers, are too lowto favor proton transfer from the matrix. In positive-ion mode, these molecules are, therefore, preferen-tially detected as cationized species (24), mostly in theform of [MþNa]þ or [MþK]þ (because some al-kali cations are ubiquitous; reaction 2.7). Given thatthe gas-phase acidities of these compounds can becomparatively high, deprotonated molecules of someof these compounds (e.g., many glycans) are morefrequently detected. Sometimes these ions are ob-served in addition to alkali-adduct ions of the form[Mþ (nþ 1)alkali ÿ nH]þ , which may even dominatethe mass spectrum. The use of matrices with partic-ularly low cation affinities like dithranol may enhancethe analyte cation yield (e.g., in the analysis of syn-thetic polymers). The latter class of compounds posesparticularly high demands on the choice of matrix,solvent system, and preparation techniques (seeChapter 11 (this volume): Synthetic Polymers).Although listed under secondary ionization mecha-nisms, reaction (2.7a) proceeds in a direct reactionbetween analyte and metal cations available in theMALDI plume and thus does not require the matrixfor the ionization, reaction (2.7b) indicates the secondpossible scenario (i.e., abtraction of a metal cationfrom a matrix-metal cation complex).

In exceptional cases, electron transfer from matrixto analyte (reaction 2.8) can yield analyte radicalions Mdþ.

Secondary reactions can also explain another classi-cal feature of MALDI: the prevalence of singly chargedions (24). For small molecules, the Coulomb repulsionprohibits the transfer of a second proton or cation toan already charged molecule. Moreover, charge reduc-tion of multiply charged ions, which can be formed byadduct ion formation with divalent metal ions likeCu2þ or Zn2þ , is energetically also favored (24).

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R. CramerUniversity of Reading, Reading, UK

K. DreisewerdUniversity of Munster, Munster, Germany

r 2007 Elsevier Ltd. All rights reserved.

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