MALDI-TOF Mass Spectrometry

** It should be noted that this is not a summary of all work involved in this ever expanding area but an overview of the actual
MALDI-TOF MS technique with some demonstrative examples from the literature of the MALDI-TOF technique. **

Characterization of polymeric materials is vital for predicting and elucidating polymer properties and morphology. Characterization typically involves: (1) molecular mass analysis utilizing gel permeation chromatography, light scattering, osmometry, or viscometry, (2) sequence of repeat units utilizing NMR spectroscopy, (3) endgroup analysis utilizing titration, NMR spectroscopy, or FT-IR spectroscopy, and (4) purity examination utilizing NMR spectroscopy, elemental analysis, and FT-IR spectroscopy. Until recently, no single technique could completely describe the above characteristics of a polymer sample. The powerful capabilities of Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry are realized with the fast and accurate determination of molar masses, the sequencing of repeat units, and recognition of polymer additives and impurities.

Mass Spectrometry in Chemical Identification

Mass Spectrometry (MS) has been appropriately used for the analysis of molar masses of molecules for the past 50 years 1. However, the application of MS to large biomolecules and synthetic polymers has been limited due to low volatility and thermal instability of these materials. These problems have been overcome to a great extent through the development of soft ionization techniques such as chemical ionization (CI) 2,3,4, secondary-ion mass spectrometry (SIMS) 2,3,5, field desorption (FD) 2,3, fast atom bombardment (FAB) 2,3, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) 6,7,8,9. The MALDI-MS technique, in particular, allows for the mass determination of large biomolecules and synthetic polymers of molar mass greater than 200,000 Daltons (Da) by ionization and vaporization without degradation.

MALDI-TOF mass spectrometry is an emerging technique offering promise for the fast and accurate determination of a number of polymer characteristics. The MALDI technique is based upon an ultraviolet absorbing matrix pioneered by Hillenkamp and Karas 6. The matrix and polymer are mixed at a molecular level in an appropriate solvent with a ~104 molar excess of the matrix. The solvent prevents aggregation of the polymer. The sample/matrix mixture is placed onto a sample probe tip. Under vacuum conditions the solvent is removed, leaving co-crystallized polymer molecules homogeneously dispersed within matrix molecules. When the pulsed laser beam is tuned to the appropriate frequency, the energy is

Figure 1: Schematic of a MALDI-TOF Mass Spectrometer

transferred to the matrix which is partially vaporized, carrying intact polymer into the vapor phase and charging the polymer chains 6. Multiple laser shots are used to improve the signal-to-noise ratio and the peak shapes, which increases the accuracy of the molar mass determination 7. In the linear TOF analyzer (drift region), the distribution of molecules emanating from a sample are imparted identical translational kinetic energies after

Figure 2: MALDI-TOF MS Sample Ionization

being subjected to the same electrical potential energy difference. These ions will then traverse the same distance down an evacuated field-free drift tube; the smaller ions arrive at the detector in a shorter amount of time than the more massive ions. Separated ion fractions arriving at the end of the drift tube are detected by an appropriate recorder that produces a signal upon impact of each ion group. The digitized data generated from successive laser shots are summed yielding a TOF mass spectrum. The TOF mass spectrum is a recording of the detector signal as a function of time. The time of flight for a molecule of mass m and charge z to travel this distance is proportional to (m/z)1/2. This relationship, t ~ (m/z)1/2, can be used to calculate the ions mass. Through calculation of the ions mass, conversion of the TOF mass spectrum to a conventional mass spectrum of mass-to-charge axis can be achieved (See figure 3).

Figure 3: Conversion from TOF spectra to Conventional Spectra

MALDI is a ‘soft’ ionization technique in which the energy from the laser is spent in volatilizing the matrix rather than in degrading the polymer. Preparation of an appropriate polymer/matrix mixture is one of the critical limiting factors for the universal application of MALDI to synthetic polymers. With the advent of MALDI in 1992, the challenge has been to discover appropriate matrix materials for use with synthetic polymers, since previous efforts were centered around biopolymers. Synthetic water-soluble polymers have been shown to be capable of analysis using similar conditions to those of biopolymers. Synthetic, organic-soluble polymers, however, have exhibited analysis complications due to their seeming incompatibility with the matrix materials. Because of this fact, only structurally simplistic synthetic water-soluble and organic-soluble polymers have been investigated to date using MALDI analysis.

The purpose of the matrix material, as alluded to previously, is two-fold: (1) absorption of energy from the laser light, thus preventing polymer decomposition, and (2) isolation of the polymer molecules from one another 7,8,9. Matrices for biopolymers have traditionally utilized just the biopolymer and the matrix material. Synthetic polymers, particularly organic-soluble polymers, have differing solubilities in the common solvents and often do not have a large concentration of ionized species. Most of the commonly used matrices are 2,5-dihydroxybenzoic acid derivatives, sinapinic acid derivatives, and indoleacrylic acid derivatives. Few compounds are useful as matrix materials due to the numerous stipulations involved: common solubility in a given solvent (water, acetonitrile, ethanol, etc.), absorption, reactivity, and volatility are conditions that must be considered before an appropriate matrix might be found for a particular synthetic polymer 7,8. In addition to the matrix material, a cationizing species is often added to increase the concentration of ionized species 10. Some linear homopolymers and condensation polymers have been shown to yield adequate spectra for analysis without a cationizing species but often alkaline salts (LiCl, NaCl, KCl) or silver trifluoroacetate have been included as the cationizing agent to increase the yield of cationized species and allow a more homogeneous cationization. Surfactants are being investigated11 for use with organic-soluble polymers where homogenization is not always possible or reproducible. Enhancement of spectra is expected where the surfactant can potentially play a dual role as both a matrix emulsifier and cationization agent.

Synthetic Polymer Analysis using MALDI

Structural investigation of homopolymers using MALDI has been limited to a discrete number of water-soluble and organic-soluble systems. MALDI studies have been shown to be applicable to polymers of a broad range of chemistry from water-soluble polymers such as poly(ethylene glycol) 12,13,17,18, poly(propylene glycol) 12, poly(styrene sulfonic acid) 14,19, and poly(acrylic acid) 14 to organic-soluble polymers such as poly(styrene) 11,12,17, and poly(butyl methacrylate) 16. Other MALDI studies in the homopolymer, condensation polymer realm have included fluorinated polymers 22, polymer blends 23, and polymer additives 24. MALDI was originally designed for the analysis of architecturally specific synthetic polymer systems such as copolymers, grafted polymers, living block copolymers, and dendrimers for which no standards exist. Montaudo et al. 25 developed statistical models for the mass spectra of the composition and microstructure of copolymers. These studies utilized principles of laser desorption (LD), fast atom bombardment (FAB), field ionization (FI), and electron impact (EI), methods in which fragmentation is observable. Actual studies utilizing MALDI analysis with copolymers have had limited investigations 26,27. Copolymers under investigation have been polybutyleneadipate-co-butylenesuccinate 27, and polyN-vinylpyrrolidone-co-vinylacetate 26. The results of copolymer studies can not be compared with other MALDI analyses since the MALDI was used strictly as a detector after polydisperse copolymers were segmented with the GPC.

Investigation of synthetic polymers utilizing MALDI techniques, first investigated by Karas et al. 10, included studies of poly(propylene glycol) (PPG) and poly(ethylene glycol) (PEG). This PPG sample was obtained from Boehringer Mannheim and used as received. Sample details are listed next to the spectra. Mixing occurs between the polymer and matrix materials on a molecular level in the solvent followed by homogeneous vacuum co-crystallization. Figure 3 shows the resultant spectra for one of the first successful MALDI analyses. The spectra is for a low molar mass (5300

Figure 4: The MALDI-TOF Spectra for Poly(Propylene Glycol)

g/mol) PPG sample. Due to the low molar mass of the polymer, the molar mass distribution is easily derived as 58 g/mol from the peak-to-peak mass increments. The molar masses obtained through MALDI, listed as Mn, Mw, and Mp, agree quite well with the value specified by the manufacturer of PPG-5300.

In another study 10, the MALDI technique was investigated for a PEG sample having a higher molar mass of 23000. At m/z 23000, the mass resolution is not sufficient to resolve adjacent oligomer molecular ions with a difference of 44 mass units. Therefore, the resultant spectra shows a convolution of the molecular ions which results in a continuous distribution. Typically, there is a limit to the peak-to-peak resolving capabilities of the MALDI which have been observed by researchers to be around 20,000 g/mol. Recently, however, a MALDI study by Montaudo et al. 13 showed samples of PEG-23,600 g/mol that had the same molecular convolution of the spectrum; however, they were able to discern the peak-to-peak mass resolution of 44 g/mol due to improved resolving power of more recently manufactured MALDI instruments. Even with the continuous distribution of the spectra, the data for the PEG-23,000 sample shows the polymer distribution is 20,000 to 25,000 g/mol, possessing a maximum of the distribution at 22930 and the centroid mass at 22950. This data still exhibits good agreement with the manufacturers value of 23,000.

Polystyrene was another system investigated by Karas et al. 10 A polystyrene sample of 20,000 g/mol from Machery & Nagel was used as received. Again, sample details are listed next to the individual spectra. Mixing between the polymer and matrix materials occurs on a molecular level in the solvent followed by homogeneous vacuum co-crystallization. Figure 4 shows the resultant spectra for MALDI analysis of poly(styrene).

In the low mass range only peaks for the matrix ions appear. This spectra has a continuous distribution due to convolution of the molecular ions, however, the molar mass for individual oligomers is apparent, as shown in the insert, with a peak-to-peak mass resolution of 104 g/mol. This sample utilized 2-nitrophenyl octyl ether, a viscous liquid as the matrix material because using 2,5-dihydroxybenzene matrixresulted in separation of the

Figure 5: A MALDI-TOF Spectra for Poly(styrene)

matrix and polymer. No results can be obtained from sample preparations with inhomogeneity between the matrix and polymer because the polymer is either directly ionized, resulting in degradation of the sample (fragmentation), or the polymer is not fully ionized, leaving ion levels below the limits-of-detection. Results from studies 11,17 in the last two years utilized a sinapinic acid derivative for the matrix material. Spectra obtained from these recent studies displayed good homogenization between the matrix and the polymer and do not show the dimer, trimer, or tetramer oligomer ion distributions. Here, the matrix material is possibly better in absorbing energy from the laser light, thus shielding the polymer from over ionization. In this poly(styrene) spectra, the dimer, trimer, and tetramer ion distributions are observed. This occurrence has been reported by other researchers 13,14,16 and does not appear to be uncommon. Agreement between the manufacturers molar mass value of 20,000 and the most probable derived from the MALDI spectra of 19740 is in good correspondence.

Certain condensation polymers have been investigated using MALDI. Contrary to most polymerization products, condensation polymers tend to possess low molar masses. However, it is the complex polymer structure of most condensation polymers that renders them interesting for MALDI studies. MALDI studies of condensation polymers have included: phenolic resins 20, epoxy resins 21,22, and polycarbonates 17,20, all of which are important technical products. Pasch et al. 20,21 studied numerous samples of the above polymers, including two commercially available oligocarbonates. MALDI samples of the two oligocarbonates, containing the matrix material of dithranol and LiCl and the oligocarbonate were mixed on a molecular level in a THF solvent solution and homogeneously vacuum co-crystallized. LiCl was added as part of the matrix to increase the formation of cationized species. Spectra A and B in Figure 5 are the resultant distributions derived from MALDI-TOF MS analysis.

Figure 6: Oligocarbonate Studies Utilizing MALDI-TOF MS

Spectra A has a straightforward molar mass distribution with peak-to-peak mass increments of 254 g/mol, equaling exactly the mass of the repeating unit of bisphenol A-based oligocarbonates. The endgroup can be derived through multiple subtraction of the repeat unit from any one of the mass peaks, as shown in figure 6. The endgroup of spectra A has therefore been derived as 228g/mol which corresponds to oligomers with hydroxy

Figure 7: A Pure Oligocarbonate Sample

endgroups. Spectra B includes the same molar mass distribution with peak-to-peak mass increments of 254 g/mol, equaling exactly the mass of the repeating unit of bisphenol A-based oligocarbonates. However, observance of further oligomeric series in the spectrum is significant because it is representative of inhomogeneity in the sample. The peaks in the oligomer series denoted by the · above the peaks are offset by 134 g/mol from the major oligomer series. It has been suggested that these oligomers contain cresol which would be present as a purposefully added chain terminator or

Figure 8: Determination of an Impurity in an Oligocarbonate

an impurity in the reaction mixture, as shown in figure 7. The peaks in the minor oligomer series denoted by the peaks with the + above them are again indicative of oligocarbonates due to the m/z 254 peak-to-peak mass increment. Endgroup calculations allow for the speculation of the two structures shown in figure 8.

Figure 9: Speculation of the Side-Reactions in the Oligocarbonate

For other valuble information check out these sites:

The USM Polymer Science Macrogalleria

General MALDI-TOF MS Information

Useage of Mass Spectrometry as a detector

Mass Spectrometry WorkGroups

PerSeptive Biosystems

University of California at San Francisco Mass Spectrometry Homepage - On line database searching.

Rockefeller : NYU Mass Spec Homepage - On line database searching; Matrix Depot.

Swiss Federal Institute of Technology Zurich Computational Biochemistry - On line database searching

Max Delruck Center Webpage - On line database searching.

National Center for Biotechnology Information - On line BLAST and Entrez searches of GenBank and dbEST.

Murrary's Mass Spectrometry Homepage - Set of hyperlinks to other mass spectrometry web sites.

Delta Mass - Extensive list of mass shifts due to amino acid modifications.

Pedro's BioMolecular Research Tools - A large collection of useful hyperlinks to mass spectrometry, protein chemistry and molecular biology sites.

American Society for Mass Spectrometry - Information on ASMS meetings and short courses.

SWISS-PROT - Annotated protein sequence database.

BIBLIOGRAPHY

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