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MALDI-MS for polymer characterization – Recent developments and future prospects
Trends in Analytical Chemistry 115 (2019) 121e128
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MALDI-MS for polymer characterization e Recent developments and future prospects a _ zon _ ski a, * Joanna Drzezd , Dagmara Jacewicz a, Alicja Sielicka b, c, Lech Chmurzyn a
Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdansk, Poland Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland c Structural Heart Research & Innovation Laboratory, Carlyle Fraser Heart Center, Division of Cardiothoracic Surgery, Emory University, Atlanta, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 11 April 2019
The development of new polymerization methods and, consequently, the generation of new polymeric materials, is associated with the development of methods for testing the properties of these materials. The matrix assisted laser desorption and ionization - mass spectrometry method (MALDI-MS) is the technique used to study various polymers. The most important advantages of this method include the ability to determine the distribution of the molecular mass of polymers, identify the components of the polymer chain, and examine the surfaces of the polymers. The investigations using MALDI-MS are conducted on different types of samples, e. g. environmental samples, synthetic polymers, colonycultured and blood-cultured samples. In this review the main recent developments of the MALDI-MS method for studies of polymers have been summarized. The future prospects in field of MALDI-MS and its application to different types of polymeric materials have been described. © 2019 Elsevier B.V. All rights reserved.
Keywords: Matrix assisted laser desorption/ionization Mass spectrometry Biopolymers Synthetic polymers Modified polymers
1. Introduction Karas et al. introduced the concept of the matrix-assisted laser desorption and ionization (MALDI) in 1985 [1]. A great achievement was the development of the ultra-fine metal plus liquid matrix method in 1988 [2]. This method improved the ionization process using the combination of a 337 nm nitrogen laser with cobalt particles with a diameter of 30 nm in glycerol. Increased interest in studies using the MALDI- mass spectrometry (MS) technique occurred in the early 90's of the twentieth century, because the first commercially available equipment for this method was introduced to the market [3]. At that time, ready-to-buy nitrogen lasers were relatively cheap, had a small size and they operated at a wavelength of 337 nm [3]. Nowadays, the MALDI-MS method is a powerful tool for the characterization of synthetic polymers and biopolymers [4]. MALDI-MS is widely used, including applications in screening and diagnostic research [4,5]. Moreover, clinical microbiology applies this technique for identification of toxins and microorganisms [5]. MALDI-MS is also used by nano-scientists, chemical analysts, clinical chemists, biomedical researchers and the communities studying
* Corresponding author. Fax: þ48 58 523 54 72. ski). E-mail address: [email protected] (L. Chmurzyn https://doi.org/10.1016/j.trac.2019.04.004 0165-9936/© 2019 Elsevier B.V. All rights reserved.
macromolecules and polymer materials. Biologists use MALDI-MS since this method has been adapted for the identification the microbes and the compounds present in the biological samples [5]. The MALDI time of flight (TOF) MS method allow to distinguish microorganisms that are very phenotypic and genotypic [6]. Over the years, detectors, matrices used for MALDI-MS, for example 2,5-dihydroxybenzoic acid and all-trans-retinoic acid, and the procedure of a sample preparation have been developed [7]. The exemplary method of the matrix optimization is the use of the high-performance liquid chromatography (HPLC) method with the reversed phase[7]. The matrix and polymer sample are placed together in the HPLC device. In the case that the retention times for the polymer and matrix are similar to each other, this ensures the best quality of MALDI-MS spectra [7,8]. The challenge for the MALDI-MS method was the analysis of insoluble polymers [9]. Skelton et al. showed that in the case of insoluble polymers such as polyamides the sample preparation via “solid/solid” method guarantees the correct MALDI-MS spectra with reproducibility and good resolution [9]. This review presents the recent developments of MALDI-MS method for studies of polymers, e. g. towards the molar mass distribution, the identification of the end group, determining the purity of the sample, the sequence in copolymers. We focus on the
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current state of the art and the future prospects in field of MALDIMS method to the analysis of biopolymers, synthetic polymers as well as modified polymers. 2. Biopolymers 2.1. Proteins The latest achievements in using of the MALDI-MS method concern the identification of bacteria, fungi and other microorganisms by the determination of various types of proteins, e.g. ribosomal proteins (Fig. 1.) [10]. Most often ribosomal, regulatory and structural proteins are determined via MALDI-MS technique [10]. In 2009 the results of the studies described by Seng et al. confirmed that MALDI-TOF-MS is a powerful tool in clinical microbiology because out of the 1660 tested samples, 84% were determined at the species level [11]. This method can replace traditional methods for identifying microbes such as Gram-staining. The main advantages of the MALDI-MS method are: wide field of applicability in relation to microorganisms, low measurement speed on the scale of seconds to minutes, and high sensitivity. MALDI-TOF-MS devices emit laser pulses with a frequency of 50 Hz, therefore the measurement time of a sample is less than 30 s [11].
Seng et al. stated that the availability of a complete database of Staphylococcus is an important element of research and the guarantee of proper identification [11]. Jo. et al. proposed the use of the MALDI-TOF-MS method together with the VITEK® 2 test [12]. In the microbiological diagnostics, the VITEK® 2 test is known as a method for the identification of microorganisms and determination of their antimicrobial susceptibility [12]. Bacteria from positive blood culture bottles have been investigated. The results of these studies showed that the value of the microorganism identification index was 81.8% on average for gram positive and negative isolates [12]. In the case of the studies on Staphylococcus and coagulase-negative Staphylococcus the MALDI-TOF-MS method combined with the VITEK® 2 test requires further improvement in order to obtain satisfactory test results. The advantage of the proposed method by Jo et al. is easy to complete and it is not expensive. Attention should be paid to the novelty of MALDI-Biotyper as a tool to identify polymer production bacteria [13]. The methylotrophs bacteria produce biopolymers e. g. polyhydroxybutyrate [13]. Moreover, MALDI, electrospray, and Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) have been combined to achieve the best possible quantitative bioanalysis [14]. In 2018 a special new matrix was designed, which allows chirality and selective detection of the structure by means of MALDI e MS [15]. In our opinion, further research into the improvement of the MALDI-TOF-MS method in the field of the identification of microorganisms is highly recommended towards inter alia MALDIBiotyper. This tool should be refined to study different types of not only bacteria but fungi producing biopolymers. MALDI-TOF-MS combined with the VITEK® 2 can be used in the near future to quickly detect and counteract epidemics even in the field of nosocomial infections. The combining these two tests be examined for the diagnosis of viruses. The combination of methods: MALDI-TOFMS and the VITEK® 2 should become a routine diagnostic method. In particular, it is important to continue research on the identification of microorganisms at the protein level by MALDI-TOF-MS because this method is cheaper than conventional methods based on immunology. 2.2. Cellulose
Fig. 1. A block diagram showing the process of identification of microorganisms by the MALDI-TOF-MS method.
In addition to proteins, polysaccharides belong to biopolymers. An example of a polysaccharide is cellulose. The lytic polysaccharide monooxygenases (LPMOs) were discovered only in 2010 and since then there has been an increase in the intensity of research on the depolymerisation of the cellulose chain [16]. The most recent studies on cellulose polymers concern the enzymes such as LPMOs damaging glycosidic bonds [16]. Quantifying and mapping the activity of LPMOs is investigated by MALDI-TOF-MS. During MALDI-TOF-MS testing of components resulting from a cut chain of cellulases, it is important to note the fact that gemdiols can be more easily dehydrated than aldonic acid. In addition, during the tests, aldonic acid occurs as a charged form and it forms double adducts. For this reason, some products of the cellulose depolymerization can be difficult to identify in the MALDI-TOF-MS spectrum [16]. The regioselectivity of LPMOs from the following fungi species Thielavia terrestris (Tt) and Thermoascus aurantiacus (Ta) from the auxiliary family of activity 9 (AA9) is investigated by MALDI-TOFMS [17]. TtAA9E and TaAA9A cut the chain of cellulose, similar oxidation. The results of the studies show that aldonic acid and 4ketoaldose are the main products of cellulose depolymerisation [17]. Unfortunately, in addition to individual products resulting from oxidative cleavage, there are also double adducts such as aldonic acid þ gemdiol, 1,5 delactone þ gemdiol and aldonic acid
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and 4-ketoaldose which exhibit the following peaks in the MALDITOF-MS spectrum where M denotes native oligosaccharide: Mþ32, Mþ14 and Mþ14, respectively [17]. Separation of the double adducts should be attempted so that they can be identified by the MALDI-MS method as single products. 2.3. Natural rubber Natural rubber is a very important polymeric material applied e. g. in the automotive industry in vulcanization [18]. Hevea brasiliensis is rubber tree where a rubber particle occurs as the specific element necessary for natural rubber storage [18]. The repeated washing is a conventional method of purifying natural rubber. In 2018 the proteome profiles of washing solutions from a rubber particle obtained after purification of natural rubber by repeated washing have been described. The Wang's et al. report showed that this way of purification leads to loss of many proteins by transferring them to the solution. It has been proven that the protein complexes revealed in the proteomic profile perform functions in a natural rubber biosynthesis. Protein determination using MALDIMS was carried out after digestion of samples by trypsin and using cyano-4-hydroxycinnamic acid as the matrix. The results of the measurements confirm the presence of 233 protein spots. This corresponds with 142 unique proteins in the washing solution from the purification process of natural rubber. Modern studies focus on the synthesis and physicochemical properties of different varieties of natural rubber, inter alia, the phosphorus version of natural rubber [19]. The MALDI-TOF-MS spectra showed the peaks corresponding with the formula [5n þ Ag]þ where n is in the range 6e29 [19]. It was possible to determine a regular pattern of ions, but it was also noticed that these peaks differ from 330 Da in the mass of the monomer. The studies carried out by Dück et al. reported for the first time the phosphorus versions of natural rubber i.e. poly (1-phospha-1,3butadiene) and poly (1-phosphaisoprene) [19]. In our opinion research should be carried out to look for ways to synthesize the arsenic version of natural rubber that will replace natural rubber since the phosphorus versions of natural rubber is known. This is important not only due to the cognitive aspect of chemistry but also because of the environment - natural rubber comes mainly from rainforests, and it is known that deforestation and climate are having a negative impact on rainforests. Furthermore, in the future, the method of purifying natural rubber should be improved in order to reduce the leaching of many proteins. 3. Synthetic polymers
Fig. 2. The molecular weight distributions of living chain of polystyrene where the black bars denote the experimental results obtained by MALDI-MS, and red line is the Poisson distribution. This figure is adapted from Ref. [20].
polyolefins. The oligomerization of 1-hexene and propene has been conducted using the following catalyst Cp0 (C5H5) ZrCl2 and Cp20 ZrCl2 where Cp0 denotes C5HMe4, C4Me4P, C5Me5, C5H4tBu, C5H3-1,3-tBu2, C5H2-1,2,4-tBu3 (Me ¼ methyl group, tBu ¼ tertiary butyl) [21]. These catalysts have been activated by methylaluminoxane (MAO). The obtained oligomers have been investigated by MALDI-TOF-MS. The results showed that the successive peaks are separated by 42 m/z and 84 m/z. These values correspond to the mass of individual monomers: propene and 1-hexen, respectively. The dispersion Q obtained by MALDI-TOF-MS equals about 1.1. The value of dispersion below 2 is the result of the insertion rate which is strictly dependent on a chain-length. The advantage of MALDI-TOF-MS is the opportunity to investigate the thermolabile molecules without fragmentation of molecular ions [21]. This method allows for the investigation of the mechanism of chain transfer. It should be noted that MALDI-TOF-MS gives good results for samples of weakly polar polymers such as polystyrene, but it is not suitable for testing polyethylene and polypropylene. For this reason, the studies on copolymers should be carried out taking into account the dependence of the percentage of weakly polar polymers embedded in the chains of nonpolar polymers in order to be able to carry out a thorough study of the MALDI-MS method.
3.1. Polyolefins
3.2. Polyethers
Polyolefins include many elastomers and thermoplastic polymers that are used in various industries e. g. for the manufacturing of plastic parts for furniture or containers. Polystyrene is a wellknown and widely used polyolefin. Oh et al. synthesized live and dead polystyrene chains via atom transfer radical polymerization [20]. Polystyrene is tested using the MALDI-MS method. This technique in this specific application allows to draw conclusions about the stereoisomerism in the chain structure [20]. The MALDIMS results showed that the molecular weight distributions of living chain of polystyrene are very close to the theoretical Poisson distribution (Fig. 2.). The Poisson distribution is a characteristic function of the probability distribution. The researchers managed to separate live chains of polystyrene from the dead chains during experiments [20]. Janiak et al. used MALDI-TOF-MS to study the 1-hexene and propene oligomers [21]. These types of oligomers belong to
Ring-opening polymerization of epoxides, ring-opening polymerization of cyclic oxanes and condensation reaction of biphenols are known methods for the synthesis of polyethers. Studies on poly (propylene glycol) have allowed, inter alia, to improve the MALDITOF-MS method inter alia in the aspect of precision at the level of 10 ppm [22]. In these studies, the ion extraction was delayed, which caused a lack of linear dependence m/z and the square of the ion flight time. The calibration procedure for MALDI-TOF-MS spectra was based on the use of signals from the two sodium cations of poly (propylene glycol) at 737.5022 (n ¼ 12) m/z and 4045.888 m/z (n ¼ 19), where n denotes the number of monomers [22]. This was the so-called double point calibration in the MALDI-TOF-MS method. In 2017, MALDI-MS was refined for testing soluble as well as insoluble polymerization products during tests of polyethers [23]. Carraher Jr et al. have synthesized organotin polyethers via the
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interfacial polycondensation [23]. For this purpose, the dicoumarol and organotin halides containing two chloride anions were used. Dicoumarol is a natural anticoagulant. When using MALDI-MS for organotins, it has some limitations because organotin is sensitive to the radiation emitted by the laser. However, for the synthesized polyethers mass spectra were recorded. MALDI-MS spectra confirm the isotopic correspondence corresponding to one tin atom. It is interesting that the results of the study show that dicoumarol does not show the fragmentation with separation carbon dioxide and methylene group as might be supposed. Recent research on the use of MALDI-MS for the polyethers studies concern the investigations of polyethylene glycol [24]. Yushkin et al. for the first time received the molecular weight cutoff using the MALDI-MS method and the polyethylene glycol chains [24]. Polyethylene glycol has been used in the linear form. MALDIMS has been used to determination of rejection coefficient values. It is the confirmation that this method increases the measurement accuracy and reliability of the obtained results. The method described by Yushkin et al. can replace the conventional popular polystyrene method. In our view, future research should focus on developing a method to reduce the sensitivity of substances such as organotins to a laser in the MALDI-MS technique. Yushkin et al. [24] applied polyethylene glycol and MALDI-MS to determine any membrane performances. Thus, the concentrations of individual components of a mixture leading to the main purpose of the determination of the molecular weight cut-off of nanofiltration membranes with using other polyethers should be investigated in the future studies. It creates perspectives for using this method in determining the usefulness of membranes in industrial-scale studies. 3.3. Polyesters Polyesters such as poly (ethylene phthalate), poly (ethylene malonate) and poly (ethylene glutarate) can be synthesized with using the enzyme lipase B from Candida Antarctica (fungi) and ultrasounds waves [25]. The use of ultrasounds for the production of polyesters is an innovative approach. The sonochemical reactions increase the efficiency during the propagation polymer chain. In the MALDI-TOF-MS studies performed by Tomke and coworkers 2,5-dihydroxy-benzoic acid was used as the matrix [25]. This method allows the exact determination of the molar mass of the obtained polyesters [25]. The distribution of molecular masses of polyesters at various polymerization stages have been subsequently determined by this method. The molecular mass resulting from the MALDI-TOF MS spectra is dependent on the degree of the polymerization process. The results show that carboxylic acid and alcohol group are located at the ends of the polyester chains [25]. It is worth noting the fact that it allows researchers to determine the suitability of polyesters in various stages of synthesis. The cross-metathesis polymerization is used for the synthesis of polyesters because it allows a strictly defined polymer sequence to be obtained [26]. The studies on the metathesis in polyesters' has gained popularity since Y. Chauvin, R. Grubbs and R. Schrock received the Nobel Prize for the metathesis in 2005. It was associated with the development of MALDI-MS method for testing polymers. The integrity of microstructural periodicity of the aliphatic polyesters obtained via the cross-metathesis polymerization is determined by MALDI-TOF-MS [26]. In the chemistry of polyesters, one should also pay attention to the innovative use of graphite as a matrix for the MALDI-MS method [27]. Interestingly, this type of the matrix has been obtained from the simple and commonly used no. 2 lead pencil [27]. The characterization of the polyesters of organotin by MALDI-MS using the graphite matrix showed two and three-part repeating
fragments of the polyester chain. The test results confirm that the polyester chain defragments in such a way that the chemical bond breaks at the site of the heteroatoms. The use of a graphite matrix shows that the MALDI-MS is developing and aims at simplifying the research method, which is very forward-looking. In our opinion, it is necessary to test graphite matrices on very sensitive polymers that are sometimes damaged during sample preparation and carrying out MALDI-MS measurements. 3.4. Polyamides Nowadays polyamides can be used as potential gas separation membranes [28]. MALDI-TOF MS allows ionene-polyamide to be characterized [28]. Polymers having an ionic group in the polymer backbone are called ionene-polymers. The studies of ionenepolyamide have been conducted using the matrix consisting of hydroxypicolinic acid and ammonium citrate. MALDI-TOF-MS testing confirmed that in the case of ionene-polyamides from 45 to 55 units are repeated in the chain [28]. Additionally, this technique has been used to study the lowviscosity composites, such as aliphatic polyamides, taking into account the distribution of the studied nylons to oligomers [29]. The analysis of MALDI-TOF-MS results confirmed the symmetric structure of oligomers. The terminal groups are not present. Photodeprotectable polyamides are studied by MALDI-TOF-MS [30]. In these studies, dithranol (1,8-dihydroxy-9 [10H]anthracenone) has been used as the matrix. In the MALDI-MS method, polymer molecules are not directly irradiated with laser light, but this process takes place by placing them in a matrix. The substance applied as a matrix is used in a corresponding excess in the relation to the amount of polymer particles, therefore the selection of a suitable matrix for testing is very important. The results of the studies showed that cyclic polymers were obtained mostly during the synthesis [30]. The obtained polymers are characterized by the fact that they have amino groups at both ends of a polymer chain. Based on the series of peaks in the MALDI-TOF-MS spectra, it is possible to determine the structure and composition of polyamides. Due to the fact that the type of groups occurring at the ends of the chain of polyamides has a very large impact on the photoprotective properties, the MALDI-TOF-MS method is ideal for analysis of polyamide structures. In the studies described in Ref. [30] the cyclic polymers belonging to polyamides were obtained mostly, thus it is necessary to develop a synthesis method that would allow the acquisition of chained polyamide bonds and the effect that it would affect the photoprotective properties of polyamides. 3.5. Copolymers The copolymerization of methyl methacrylate (MMA) with 1octene has been studied by MALDI-TOF-MS [31]. The atom transfer radical (co)polymerization (ATRP) has been investigated and it allows the narrow molar mass distributions of the synthesized copolymers to be obtained [31]. For this type copolymers i.e. methyl methacrylate (MMA) and 1-octene, the MALDI-TOF-MS studies have been performed using (trans-2-[3-(4-tert-butylphenyl)-2methyl-2-propenylidene]malononitrile as the matrix. The calculations based on the MALDI-TOF-MS spectra showed that the fragments of the copolymer chain occurred in the cationic form with potassium [31]. In addition, the copolymers obtained as a result of living anionic copolymerization were also tested with the MALDITOF-MS technique (Fig. 3.) [32]. The samples of the obtained copolymers of butadiene and diphenylethylene were dissolved in
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Fig. 3. The MALDI-TOF-MS results of the studies of the copolymers obtained via living anionic copolymerization. The figure is adapted from Ref. [32].
tetrahydrofuran (THF) or chloroform. The copolymer concentration was equal to 1 mg/mL. Hutchings et al. revealed that the MALDITOF-MS method is a powerful technique in the study of sequences in the copolymer chain. Based on the analysis of the MALDI-TOF-MS spectrum and performed calculations, it is determined exactly which the chemical compounds participating in the copolymerization reaction are at the ends of the copolymer chain. In 2017, MALDI-spiral-TOF-MS combined with supercritical methanolysis has been applied to study the acrylic copolymer [33]. Recent results of the studies on the MALDI-MS technique show the innovative approach in the determination of the composition and the structure of the copolymers [34]. 2 (-4-Hydroxyphenylazo-) benzoic acid has been used as the MALDI-matrix. The statistical model of mass analysis of copolymers (MACO) has been applied to perform calculations leading to the solution of the complex MALDITOF-MS spectrum. The task was difficult since the difference between successive units was 1 g/mol. The studies were conducted for peaks within the region of 1000e3000 m/z [34]. The MACO calculation program is extremely useful because it eliminates the problem of multiple peaks assignment. The calculations in the MACO program are carried out for a large number of combinations taking into account the sequence of the copolymer chain and the groups found at each end of the chain. The resolution of the spectra obtained using the MALDI-TOF-MS technique depends on the method of data reception and recording. In the case of copolymers, more complicated MALDI-MS spectra are obtained compared to homopolymers. Taking into account the collection of data during the measurement and their subsequent analysis, the spectra of copolymers are more demanding than in the case of spectra of other homogeneous polymers. The method of collecting data in the MALDI-TOF-MS method is very important for the quality of the obtained spectra. MALDI-TOF-MS gives more accurate results in determining the groups present at each end of the copolymer compared to other techniques for characterizing polymers such as nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). In our opinion, additional research is needed to test the MACO program on oligomers within the region of 300e1000 m/z, because in this case MACO will be more universal for testing.
medium of reaction. The process occurs in the presence of zinc(II) chloride or sulfuric acid acting as the catalyst. Currently, the synthetic polymeric materials containing built-in cellulose acetate into the main chain is the aim of the studies [35]. For this purpose, the 1,2-intermolecular radical addition method (1,2-IRA) is used. Moreira et al. synthesized the copolymers containing cellulose acetate and styrene [35]. The characterization of the properties of the obtained polymers is carried out using the MALDI-MS method. The materials containing cellulose acetate are used for the production of various elements that adversely affect the environment, e.g. cigarette filters [35]. Thus, the studies on enzymes that degrade modified cellulose are very popular [36]. LPMOs cause the cellulose acetate degradation in the same way as the degradation of cellulose [16,36]. In the case of a polymer chain containing cellulose acetate, the degradation takes place on the backbone of the chain and on the acetyl group of the polymer [36]. 4.2. Modified starch Ren et al. obtained the modified starch using 1,4-a-glucan branching enzyme (GBE) and tapioca starch [37]. The starch from tapioca has resistant starch which reduces the risk of diseases resulting from an incorrect diet. For this reason, the investigations
Table 1 Collection the main recent developments of MALDI-MS method including the type of polymer sample. The type of polymer sample
The main recent developments of MALDI-MS method
biopolymer
the laser pulses with a frequency of 50 Hz through the use of nitrogen lasers and by this the measurement time of a sample is less than 30 s [11] the measurement time of a sample is less than 30 s [11] the combination of the MALDI-TOF-MS method with the VITEK® 2 test is used in the diagnosis of microorganisms [12] the analysis of the thermolabile molecules without fragmentation molecular ions [21] the precise determination of sequences in the copolymer chain [32] the use of the MACO program for the polymer chain analysis [34] the characterization of a highly branched structure of modified starch [38] the precise determination of the sites of chain degradation of polymers containing cellulose acetate [36]
synthetic polymer
4. Modified polymers 4.1. Modified cellulose Cellulose acetate belongs to the group of modified polymers. Cellulose acetate is obtained as the result of the acetylation reaction of cellulose with acetic anhydride in glacial acetic acid as the
modified polymer
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Table 2 Comparison of MS techniques for analysis of polymers. Method
Major advantages
Major disadvantages
Electrospray ionization - Mass Spectrometry (ESI - MS)
handling samples with large masses [41] the ability to analyze biological samples in which non-covalent interactions occur [41]
Liquid Secondary Ion Mass Spectrometry (LSIMS)
no gas load during measurements (lower noise) [42] easy spraying of high weight ions [42] high sensitivity [43] multidimensional method of studies [43] automation [43] low sample volumes [43] low cost [43] automation [43] high resolution [43] sensitivity from millimolar to nanomolar [43] separation of isobaric cooligomers [43] studies of thermally labile compounds [43]
the results obtained for the mixtures are unreliable [41] many charges occurring with molecular ions result in a lack of data transparency [41] occurrence of mass interferences [42] dependence of secondary ions on the matrix [42] restricted mass range of studied samples [43] sensitive to interfering compounds [43]
Liquid chromatography - Mass Spectrometry (LC - MS) Capillary electrophoresisemass spectrometry (CE - MS) Gas Chromatography - Mass Spectrometry (GC - MS) High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC/ ESI-MS) Inductively Coupled Plasma - Mass Spectrometry (ICP - MS)
excellent detection limits [44] powerful semiquantitative analysis [44] wide dynamic range [44]
on starch as well as modified starch are very important. MALDITOF-MS is a useful method to determine the molecular mass distribution of the modified starch. 2,5-Dihydroxybenzoic acid is used as the matrix in these studies. The results of the studies show that the peaks corresponding with m/z can be equal to the molecular weight because the molecule has the one positive charge. Moreover, modified starch is also obtained by the use of a thermostable branched enzyme from Rhodothermus obamensis and amylomaltase from Thermus thermophiles [38]. The aim of the studies was to obtain modified starch with a highly branched structure. MALDITOF-MS has been used to investigate the molecular mass distribution of the obtained products of modified starch. The spectra contained peaks above the value 4000 m/z [38]. Moreover, in these studies the formation of small cyclodextrins was examined by MALDI-TOF-MS. The smallest identified cyclodextrin was the gcyclodextrin present in the spectra at 1320 m/z. The low values of Molecular weight [Mw] allow formation of cyclostructures during the reaction, such as inter alia cyclo-amylose and the clusters of cyclo-amylopectin [38]. In the case of studies on modified polymers, it should be kept in mind that the amount of modified polymer sample needed to carry out the MALDI-MS study is the relative content of the polymer in the test sample, and it is not the absolute amount of modified polymer in the specified solvent. Obtaining a calibration curve using identical samples of modified polymers is impossible due to the heterogeneity and polydispersity of polymers. In order to determine the content of precursor polymer in the sample of the obtained modified polymer, the method combining the use of an internal standard and the addition of a standard substance is used. Future research on MALDI-MS development should focus on solving the problem of signal attenuation in complex mixtures, e. g. containing samples of various modified polymers mainly modified cellulose and modified starch. On the other hand, it is also possible to refine the method of decomposition of modified starch so that complicated mixtures of the cyclodextrins are not formed, and the problem of signal attenuation could be avoided. 5. Data imaging and data processing in MALDI MALDI imaging is subject to continuous improvement, thanks to the new techniques currently used, such as 3D MALDI-imaging,
not suited for protein characterized by a weight larger than 20 kD [43]
slow analysis time [43] limited number of analyzed molecules [43] sensitive to salts [43] studies on only polar sample [43] complexity of method [43]
argide and matrix interferences, polyatomic interference [44] plasma tail in light path [44]
high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR) and MALDI-Orbitrap-imaging [39]. MALDIFTICR and 3D MALDI-imaging are often used to investigations of biopolymers and metabolites. 3D MALDI-imaging is the method without marking [39]. It is recommended to use magnetic resonance imaging in combination with 3D MALDI-imaging in order to improve the interpretation of MALDI images [39]. Modern research proves that MALDI-Orbitrap-imaging allow to a quick and easy analysis. Moreover, the advantage of this method is a miniature design. Data processing in MALDI-MS consists of two stages - first, preprocessing and secondly statistical analysis [40]. The spectra are subjected to the following rotation: the removal of the background, normalization of intensity, and alignment [40]. The simpler the preprocessing method, the further processing of data is faster. The availability and diversity of data for analysis using the MALDI-MS method is constantly increasing, but there is a problem with the reduction of measurement errors. 6. Conclusions This review shows that the MALDI-MS technique is a powerful analytical tool to study and characterize not only synthetic polymers but also polymers obtained from natural sources. The solvent is not required in the MALDI-MS method; therefore optimizations of this method can eliminate this dependence. In contrast, the image of spectra obtained by this method is dependent on the matrix and the method of preparing the sample for measurements. The main recent developments of MALDI-MS method for studies of polymers have been summarized in Table 1. Moreover, the comparison of MALDI-MS with other MS techniques is presented in Table 2. The future prospects in field of MALDI-MS method to different types of polymeric materials have been described in detail. Acquiring new polymeric materials is the subject of contemporary research [45e47]. There are ongoing studies on new catalysts of polymerization reactions, in particular on non-metallocene complex compounds containing Cr3þ and V4þ cations [48e57]. The results of the studies on the catalytic activity of these complex compounds are promising. The synthesis of new polymeric
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materials consequently brings with it the development of the MALDI-MS technique for the characterization and analysis of polymers [34,58]. We predict that in the near future it will be possible to further explore the structure of polymers and the obtained MALDI-MS spectra will be even higher resolution than those present due to the innovative approach to sample analysis inter alia through computer techniques and simulations associated with it. Acknowledgements This work was supported by the National Science Centre, Poland under Grant number 2015/19/N/ST5/00276. References [1] M. Karas, D. Bachmann, F. Hillenkamp, Influence of the wavelength in highirradiance ultraviolet laser desorption mass spectrometry of organic molecules, Anal. Chem. 57 (1985) 2935e2939. https://doi.org/10.1021/ac00291a042. [2] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, T. Matsuo, Protein and polymer analyses up to m/z 100 000 by laser ionization timeofflight mass spectrometry, Rapid Commun. Mass Spectrom. 2 (1988) 151e153. https://doi. org/10.1002/rcm.1290020802. [3] M. Karas, U. Bahr, Laser desorption ionization mass spectrometry of large biomolecules, Trac. Trends Anal. Chem. 9 (1990) 321e325. https://doi.org/10. 1016/0165-9936(90)85065-F. [4] F. Hillenkamp, J. Peter-Katalinic (Editors), MALDI MS: a Practical Guide to Instrumentation, Methods and Applications, second ed., John Wiley & Sons, Hoboken (New Jersey), 2013. [5] W. Pusch, M. Kostrzewa, Application of MALDI-TOF mass spectrometry in screening and diagnostic research, Curr. Pharmaceut. Des. 11 (2005) 2577e2591. https://doi.org/10.2174/1381612054546932. [6] T.R. Sandrin, J.E. Goldstein, S. Schumaker, MALDI TOF MS profiling of bacteria at the strain level: a review, Mass Spectrom. Rev. 32 (2013) 188e217. https:// doi.org/10.1002/mas.21359. [7] G. Montaudo, F. Samperi, M.S. Montaudo, Characterization of synthetic polymers by MALDI-MS, Prog. Polym. Sci. 31 (2006) 277e357. https://doi.org/10. 1016/j.progpolymsci.2005.12.001. [8] A.J. Hoteling, W.J. Erb, R. Tyson, K.G. Owens, Exploring the importance of the relative solubility of matrix and analyte in MALDI sample preparation using HPLC, Anal. Chem. 76 (2004) 5157e5164. https://doi.org/10.1021/ac049566m. [9] R. Skelton, F. Dubois, R. Zenobi, A MALDI sample preparation method suitable for insoluble polymers, Anal. Chem. 72 (2000) 1707e1710. https://doi.org/10. 1021/ac991181u. [10] S. Angeletti, Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology, J. Microbiol. Methods 138 (2017) 20e29. https://doi.org/10.1016/j.mimet.2016.09.003. [11] P. Seng, M. Drancourt, F. Gouriet, B. La Scola, P.E. Fournier, J.M. Rolain, D. Raoult, Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Clin. Infect. Dis. 49 (2009) 543e551. https://doi.org/10.1086/ 600885. [12] S.J. Jo, K.G. Park, K. Han, D.J. Park, Y.J. Park, Direct identification and antimicrobial susceptibility testing of bacteria from positive blood culture bottles by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Vitek 2 system, Ann. Lab. Med. 36 (2016) 117e123. https://doi.org/10. 3343/alm.2016.36.2.117. [13] B. Karolski, L.O. Cardoso, L.H. Gracioso, C.A. Nascimento, E.A. Perpetuo, MALDIBiotyper as a tool to identify polymer producer bacteria, J. Microbiol. Methods 153 (2018) 127e132. https://doi.org/10.1016/j.mimet.2018.09.016. [14] M.W. Linscheid, Molecules and elements for quantitative bioanalysis: the allure of using electrospray, MALDI, and ICP mass spectrometry sidebyside, Mass Spectrom. Rev. 38 (2019) 169e186. https://doi.org/10.1002/mas.21567. [15] Y. Fujii, J. Xu, T. Fujino, Chirality and structure-selective MALDI using mold matrix, Sci. Rep. 8 (2018) 13138. https://doi.org/10.1038/s41598-018-31629-6. [16] B. Westereng, J.S. Loose, G. Vaaje-Kolstad, F.L. Aachmann, M. Sørlie, V.G. Eijsink, Analytical tools for characterizing cellulose-active lytic polysaccharide monooxygenases (LPMOs), in: M. Lübeck (Editor), Cellulases. Methods in Molecular Biology, Humana Press, New York, 2018, pp. 219e246. https://doi.org/10.1007/978-1-4939-7877-9_16. [17] I.J. Kim, N. Seo, H.J. An, J.H. Kim, P.V. Harris, K.H. Kim, Type-dependent action modes of Tt AA9E and Ta AA9A acting on cellulose and differently pretreated lignocellulosic substrates, Biotechnol. Biofuels 10 (2017) 46. https://doi.org/ 10.1186/s13068-017-0721-4. [18] D. Wang, Y. Sun, L. Chang, Z. Tong, Q. Xie, X. Jin, L. Zhu, P. He, H. Li, X. Wang, Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis, J. Proteomics 182 (2018) 53e64. https://doi.org/10.1016/j.jprot. 2018.05.002.
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MALDI-MS for polymer characterization e Recent developments and future prospects a _ zon _ ski a, * Joanna Drzezd , Dagmara Jacewicz a, Alicja Sielicka b, c, Lech Chmurzyn a
Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdansk, Poland Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland c Structural Heart Research & Innovation Laboratory, Carlyle Fraser Heart Center, Division of Cardiothoracic Surgery, Emory University, Atlanta, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 11 April 2019
The development of new polymerization methods and, consequently, the generation of new polymeric materials, is associated with the development of methods for testing the properties of these materials. The matrix assisted laser desorption and ionization - mass spectrometry method (MALDI-MS) is the technique used to study various polymers. The most important advantages of this method include the ability to determine the distribution of the molecular mass of polymers, identify the components of the polymer chain, and examine the surfaces of the polymers. The investigations using MALDI-MS are conducted on different types of samples, e. g. environmental samples, synthetic polymers, colonycultured and blood-cultured samples. In this review the main recent developments of the MALDI-MS method for studies of polymers have been summarized. The future prospects in field of MALDI-MS and its application to different types of polymeric materials have been described. © 2019 Elsevier B.V. All rights reserved.
Keywords: Matrix assisted laser desorption/ionization Mass spectrometry Biopolymers Synthetic polymers Modified polymers
1. Introduction Karas et al. introduced the concept of the matrix-assisted laser desorption and ionization (MALDI) in 1985 [1]. A great achievement was the development of the ultra-fine metal plus liquid matrix method in 1988 [2]. This method improved the ionization process using the combination of a 337 nm nitrogen laser with cobalt particles with a diameter of 30 nm in glycerol. Increased interest in studies using the MALDI- mass spectrometry (MS) technique occurred in the early 90's of the twentieth century, because the first commercially available equipment for this method was introduced to the market [3]. At that time, ready-to-buy nitrogen lasers were relatively cheap, had a small size and they operated at a wavelength of 337 nm [3]. Nowadays, the MALDI-MS method is a powerful tool for the characterization of synthetic polymers and biopolymers [4]. MALDI-MS is widely used, including applications in screening and diagnostic research [4,5]. Moreover, clinical microbiology applies this technique for identification of toxins and microorganisms [5]. MALDI-MS is also used by nano-scientists, chemical analysts, clinical chemists, biomedical researchers and the communities studying
* Corresponding author. Fax: þ48 58 523 54 72. ski). E-mail address: [email protected] (L. Chmurzyn https://doi.org/10.1016/j.trac.2019.04.004 0165-9936/© 2019 Elsevier B.V. All rights reserved.
macromolecules and polymer materials. Biologists use MALDI-MS since this method has been adapted for the identification the microbes and the compounds present in the biological samples [5]. The MALDI time of flight (TOF) MS method allow to distinguish microorganisms that are very phenotypic and genotypic [6]. Over the years, detectors, matrices used for MALDI-MS, for example 2,5-dihydroxybenzoic acid and all-trans-retinoic acid, and the procedure of a sample preparation have been developed [7]. The exemplary method of the matrix optimization is the use of the high-performance liquid chromatography (HPLC) method with the reversed phase[7]. The matrix and polymer sample are placed together in the HPLC device. In the case that the retention times for the polymer and matrix are similar to each other, this ensures the best quality of MALDI-MS spectra [7,8]. The challenge for the MALDI-MS method was the analysis of insoluble polymers [9]. Skelton et al. showed that in the case of insoluble polymers such as polyamides the sample preparation via “solid/solid” method guarantees the correct MALDI-MS spectra with reproducibility and good resolution [9]. This review presents the recent developments of MALDI-MS method for studies of polymers, e. g. towards the molar mass distribution, the identification of the end group, determining the purity of the sample, the sequence in copolymers. We focus on the
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current state of the art and the future prospects in field of MALDIMS method to the analysis of biopolymers, synthetic polymers as well as modified polymers. 2. Biopolymers 2.1. Proteins The latest achievements in using of the MALDI-MS method concern the identification of bacteria, fungi and other microorganisms by the determination of various types of proteins, e.g. ribosomal proteins (Fig. 1.) [10]. Most often ribosomal, regulatory and structural proteins are determined via MALDI-MS technique [10]. In 2009 the results of the studies described by Seng et al. confirmed that MALDI-TOF-MS is a powerful tool in clinical microbiology because out of the 1660 tested samples, 84% were determined at the species level [11]. This method can replace traditional methods for identifying microbes such as Gram-staining. The main advantages of the MALDI-MS method are: wide field of applicability in relation to microorganisms, low measurement speed on the scale of seconds to minutes, and high sensitivity. MALDI-TOF-MS devices emit laser pulses with a frequency of 50 Hz, therefore the measurement time of a sample is less than 30 s [11].
Seng et al. stated that the availability of a complete database of Staphylococcus is an important element of research and the guarantee of proper identification [11]. Jo. et al. proposed the use of the MALDI-TOF-MS method together with the VITEK® 2 test [12]. In the microbiological diagnostics, the VITEK® 2 test is known as a method for the identification of microorganisms and determination of their antimicrobial susceptibility [12]. Bacteria from positive blood culture bottles have been investigated. The results of these studies showed that the value of the microorganism identification index was 81.8% on average for gram positive and negative isolates [12]. In the case of the studies on Staphylococcus and coagulase-negative Staphylococcus the MALDI-TOF-MS method combined with the VITEK® 2 test requires further improvement in order to obtain satisfactory test results. The advantage of the proposed method by Jo et al. is easy to complete and it is not expensive. Attention should be paid to the novelty of MALDI-Biotyper as a tool to identify polymer production bacteria [13]. The methylotrophs bacteria produce biopolymers e. g. polyhydroxybutyrate [13]. Moreover, MALDI, electrospray, and Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) have been combined to achieve the best possible quantitative bioanalysis [14]. In 2018 a special new matrix was designed, which allows chirality and selective detection of the structure by means of MALDI e MS [15]. In our opinion, further research into the improvement of the MALDI-TOF-MS method in the field of the identification of microorganisms is highly recommended towards inter alia MALDIBiotyper. This tool should be refined to study different types of not only bacteria but fungi producing biopolymers. MALDI-TOF-MS combined with the VITEK® 2 can be used in the near future to quickly detect and counteract epidemics even in the field of nosocomial infections. The combining these two tests be examined for the diagnosis of viruses. The combination of methods: MALDI-TOFMS and the VITEK® 2 should become a routine diagnostic method. In particular, it is important to continue research on the identification of microorganisms at the protein level by MALDI-TOF-MS because this method is cheaper than conventional methods based on immunology. 2.2. Cellulose
Fig. 1. A block diagram showing the process of identification of microorganisms by the MALDI-TOF-MS method.
In addition to proteins, polysaccharides belong to biopolymers. An example of a polysaccharide is cellulose. The lytic polysaccharide monooxygenases (LPMOs) were discovered only in 2010 and since then there has been an increase in the intensity of research on the depolymerisation of the cellulose chain [16]. The most recent studies on cellulose polymers concern the enzymes such as LPMOs damaging glycosidic bonds [16]. Quantifying and mapping the activity of LPMOs is investigated by MALDI-TOF-MS. During MALDI-TOF-MS testing of components resulting from a cut chain of cellulases, it is important to note the fact that gemdiols can be more easily dehydrated than aldonic acid. In addition, during the tests, aldonic acid occurs as a charged form and it forms double adducts. For this reason, some products of the cellulose depolymerization can be difficult to identify in the MALDI-TOF-MS spectrum [16]. The regioselectivity of LPMOs from the following fungi species Thielavia terrestris (Tt) and Thermoascus aurantiacus (Ta) from the auxiliary family of activity 9 (AA9) is investigated by MALDI-TOFMS [17]. TtAA9E and TaAA9A cut the chain of cellulose, similar oxidation. The results of the studies show that aldonic acid and 4ketoaldose are the main products of cellulose depolymerisation [17]. Unfortunately, in addition to individual products resulting from oxidative cleavage, there are also double adducts such as aldonic acid þ gemdiol, 1,5 delactone þ gemdiol and aldonic acid
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and 4-ketoaldose which exhibit the following peaks in the MALDITOF-MS spectrum where M denotes native oligosaccharide: Mþ32, Mþ14 and Mþ14, respectively [17]. Separation of the double adducts should be attempted so that they can be identified by the MALDI-MS method as single products. 2.3. Natural rubber Natural rubber is a very important polymeric material applied e. g. in the automotive industry in vulcanization [18]. Hevea brasiliensis is rubber tree where a rubber particle occurs as the specific element necessary for natural rubber storage [18]. The repeated washing is a conventional method of purifying natural rubber. In 2018 the proteome profiles of washing solutions from a rubber particle obtained after purification of natural rubber by repeated washing have been described. The Wang's et al. report showed that this way of purification leads to loss of many proteins by transferring them to the solution. It has been proven that the protein complexes revealed in the proteomic profile perform functions in a natural rubber biosynthesis. Protein determination using MALDIMS was carried out after digestion of samples by trypsin and using cyano-4-hydroxycinnamic acid as the matrix. The results of the measurements confirm the presence of 233 protein spots. This corresponds with 142 unique proteins in the washing solution from the purification process of natural rubber. Modern studies focus on the synthesis and physicochemical properties of different varieties of natural rubber, inter alia, the phosphorus version of natural rubber [19]. The MALDI-TOF-MS spectra showed the peaks corresponding with the formula [5n þ Ag]þ where n is in the range 6e29 [19]. It was possible to determine a regular pattern of ions, but it was also noticed that these peaks differ from 330 Da in the mass of the monomer. The studies carried out by Dück et al. reported for the first time the phosphorus versions of natural rubber i.e. poly (1-phospha-1,3butadiene) and poly (1-phosphaisoprene) [19]. In our opinion research should be carried out to look for ways to synthesize the arsenic version of natural rubber that will replace natural rubber since the phosphorus versions of natural rubber is known. This is important not only due to the cognitive aspect of chemistry but also because of the environment - natural rubber comes mainly from rainforests, and it is known that deforestation and climate are having a negative impact on rainforests. Furthermore, in the future, the method of purifying natural rubber should be improved in order to reduce the leaching of many proteins. 3. Synthetic polymers
Fig. 2. The molecular weight distributions of living chain of polystyrene where the black bars denote the experimental results obtained by MALDI-MS, and red line is the Poisson distribution. This figure is adapted from Ref. [20].
polyolefins. The oligomerization of 1-hexene and propene has been conducted using the following catalyst Cp0 (C5H5) ZrCl2 and Cp20 ZrCl2 where Cp0 denotes C5HMe4, C4Me4P, C5Me5, C5H4tBu, C5H3-1,3-tBu2, C5H2-1,2,4-tBu3 (Me ¼ methyl group, tBu ¼ tertiary butyl) [21]. These catalysts have been activated by methylaluminoxane (MAO). The obtained oligomers have been investigated by MALDI-TOF-MS. The results showed that the successive peaks are separated by 42 m/z and 84 m/z. These values correspond to the mass of individual monomers: propene and 1-hexen, respectively. The dispersion Q obtained by MALDI-TOF-MS equals about 1.1. The value of dispersion below 2 is the result of the insertion rate which is strictly dependent on a chain-length. The advantage of MALDI-TOF-MS is the opportunity to investigate the thermolabile molecules without fragmentation of molecular ions [21]. This method allows for the investigation of the mechanism of chain transfer. It should be noted that MALDI-TOF-MS gives good results for samples of weakly polar polymers such as polystyrene, but it is not suitable for testing polyethylene and polypropylene. For this reason, the studies on copolymers should be carried out taking into account the dependence of the percentage of weakly polar polymers embedded in the chains of nonpolar polymers in order to be able to carry out a thorough study of the MALDI-MS method.
3.1. Polyolefins
3.2. Polyethers
Polyolefins include many elastomers and thermoplastic polymers that are used in various industries e. g. for the manufacturing of plastic parts for furniture or containers. Polystyrene is a wellknown and widely used polyolefin. Oh et al. synthesized live and dead polystyrene chains via atom transfer radical polymerization [20]. Polystyrene is tested using the MALDI-MS method. This technique in this specific application allows to draw conclusions about the stereoisomerism in the chain structure [20]. The MALDIMS results showed that the molecular weight distributions of living chain of polystyrene are very close to the theoretical Poisson distribution (Fig. 2.). The Poisson distribution is a characteristic function of the probability distribution. The researchers managed to separate live chains of polystyrene from the dead chains during experiments [20]. Janiak et al. used MALDI-TOF-MS to study the 1-hexene and propene oligomers [21]. These types of oligomers belong to
Ring-opening polymerization of epoxides, ring-opening polymerization of cyclic oxanes and condensation reaction of biphenols are known methods for the synthesis of polyethers. Studies on poly (propylene glycol) have allowed, inter alia, to improve the MALDITOF-MS method inter alia in the aspect of precision at the level of 10 ppm [22]. In these studies, the ion extraction was delayed, which caused a lack of linear dependence m/z and the square of the ion flight time. The calibration procedure for MALDI-TOF-MS spectra was based on the use of signals from the two sodium cations of poly (propylene glycol) at 737.5022 (n ¼ 12) m/z and 4045.888 m/z (n ¼ 19), where n denotes the number of monomers [22]. This was the so-called double point calibration in the MALDI-TOF-MS method. In 2017, MALDI-MS was refined for testing soluble as well as insoluble polymerization products during tests of polyethers [23]. Carraher Jr et al. have synthesized organotin polyethers via the
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interfacial polycondensation [23]. For this purpose, the dicoumarol and organotin halides containing two chloride anions were used. Dicoumarol is a natural anticoagulant. When using MALDI-MS for organotins, it has some limitations because organotin is sensitive to the radiation emitted by the laser. However, for the synthesized polyethers mass spectra were recorded. MALDI-MS spectra confirm the isotopic correspondence corresponding to one tin atom. It is interesting that the results of the study show that dicoumarol does not show the fragmentation with separation carbon dioxide and methylene group as might be supposed. Recent research on the use of MALDI-MS for the polyethers studies concern the investigations of polyethylene glycol [24]. Yushkin et al. for the first time received the molecular weight cutoff using the MALDI-MS method and the polyethylene glycol chains [24]. Polyethylene glycol has been used in the linear form. MALDIMS has been used to determination of rejection coefficient values. It is the confirmation that this method increases the measurement accuracy and reliability of the obtained results. The method described by Yushkin et al. can replace the conventional popular polystyrene method. In our view, future research should focus on developing a method to reduce the sensitivity of substances such as organotins to a laser in the MALDI-MS technique. Yushkin et al. [24] applied polyethylene glycol and MALDI-MS to determine any membrane performances. Thus, the concentrations of individual components of a mixture leading to the main purpose of the determination of the molecular weight cut-off of nanofiltration membranes with using other polyethers should be investigated in the future studies. It creates perspectives for using this method in determining the usefulness of membranes in industrial-scale studies. 3.3. Polyesters Polyesters such as poly (ethylene phthalate), poly (ethylene malonate) and poly (ethylene glutarate) can be synthesized with using the enzyme lipase B from Candida Antarctica (fungi) and ultrasounds waves [25]. The use of ultrasounds for the production of polyesters is an innovative approach. The sonochemical reactions increase the efficiency during the propagation polymer chain. In the MALDI-TOF-MS studies performed by Tomke and coworkers 2,5-dihydroxy-benzoic acid was used as the matrix [25]. This method allows the exact determination of the molar mass of the obtained polyesters [25]. The distribution of molecular masses of polyesters at various polymerization stages have been subsequently determined by this method. The molecular mass resulting from the MALDI-TOF MS spectra is dependent on the degree of the polymerization process. The results show that carboxylic acid and alcohol group are located at the ends of the polyester chains [25]. It is worth noting the fact that it allows researchers to determine the suitability of polyesters in various stages of synthesis. The cross-metathesis polymerization is used for the synthesis of polyesters because it allows a strictly defined polymer sequence to be obtained [26]. The studies on the metathesis in polyesters' has gained popularity since Y. Chauvin, R. Grubbs and R. Schrock received the Nobel Prize for the metathesis in 2005. It was associated with the development of MALDI-MS method for testing polymers. The integrity of microstructural periodicity of the aliphatic polyesters obtained via the cross-metathesis polymerization is determined by MALDI-TOF-MS [26]. In the chemistry of polyesters, one should also pay attention to the innovative use of graphite as a matrix for the MALDI-MS method [27]. Interestingly, this type of the matrix has been obtained from the simple and commonly used no. 2 lead pencil [27]. The characterization of the polyesters of organotin by MALDI-MS using the graphite matrix showed two and three-part repeating
fragments of the polyester chain. The test results confirm that the polyester chain defragments in such a way that the chemical bond breaks at the site of the heteroatoms. The use of a graphite matrix shows that the MALDI-MS is developing and aims at simplifying the research method, which is very forward-looking. In our opinion, it is necessary to test graphite matrices on very sensitive polymers that are sometimes damaged during sample preparation and carrying out MALDI-MS measurements. 3.4. Polyamides Nowadays polyamides can be used as potential gas separation membranes [28]. MALDI-TOF MS allows ionene-polyamide to be characterized [28]. Polymers having an ionic group in the polymer backbone are called ionene-polymers. The studies of ionenepolyamide have been conducted using the matrix consisting of hydroxypicolinic acid and ammonium citrate. MALDI-TOF-MS testing confirmed that in the case of ionene-polyamides from 45 to 55 units are repeated in the chain [28]. Additionally, this technique has been used to study the lowviscosity composites, such as aliphatic polyamides, taking into account the distribution of the studied nylons to oligomers [29]. The analysis of MALDI-TOF-MS results confirmed the symmetric structure of oligomers. The terminal groups are not present. Photodeprotectable polyamides are studied by MALDI-TOF-MS [30]. In these studies, dithranol (1,8-dihydroxy-9 [10H]anthracenone) has been used as the matrix. In the MALDI-MS method, polymer molecules are not directly irradiated with laser light, but this process takes place by placing them in a matrix. The substance applied as a matrix is used in a corresponding excess in the relation to the amount of polymer particles, therefore the selection of a suitable matrix for testing is very important. The results of the studies showed that cyclic polymers were obtained mostly during the synthesis [30]. The obtained polymers are characterized by the fact that they have amino groups at both ends of a polymer chain. Based on the series of peaks in the MALDI-TOF-MS spectra, it is possible to determine the structure and composition of polyamides. Due to the fact that the type of groups occurring at the ends of the chain of polyamides has a very large impact on the photoprotective properties, the MALDI-TOF-MS method is ideal for analysis of polyamide structures. In the studies described in Ref. [30] the cyclic polymers belonging to polyamides were obtained mostly, thus it is necessary to develop a synthesis method that would allow the acquisition of chained polyamide bonds and the effect that it would affect the photoprotective properties of polyamides. 3.5. Copolymers The copolymerization of methyl methacrylate (MMA) with 1octene has been studied by MALDI-TOF-MS [31]. The atom transfer radical (co)polymerization (ATRP) has been investigated and it allows the narrow molar mass distributions of the synthesized copolymers to be obtained [31]. For this type copolymers i.e. methyl methacrylate (MMA) and 1-octene, the MALDI-TOF-MS studies have been performed using (trans-2-[3-(4-tert-butylphenyl)-2methyl-2-propenylidene]malononitrile as the matrix. The calculations based on the MALDI-TOF-MS spectra showed that the fragments of the copolymer chain occurred in the cationic form with potassium [31]. In addition, the copolymers obtained as a result of living anionic copolymerization were also tested with the MALDITOF-MS technique (Fig. 3.) [32]. The samples of the obtained copolymers of butadiene and diphenylethylene were dissolved in
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Fig. 3. The MALDI-TOF-MS results of the studies of the copolymers obtained via living anionic copolymerization. The figure is adapted from Ref. [32].
tetrahydrofuran (THF) or chloroform. The copolymer concentration was equal to 1 mg/mL. Hutchings et al. revealed that the MALDITOF-MS method is a powerful technique in the study of sequences in the copolymer chain. Based on the analysis of the MALDI-TOF-MS spectrum and performed calculations, it is determined exactly which the chemical compounds participating in the copolymerization reaction are at the ends of the copolymer chain. In 2017, MALDI-spiral-TOF-MS combined with supercritical methanolysis has been applied to study the acrylic copolymer [33]. Recent results of the studies on the MALDI-MS technique show the innovative approach in the determination of the composition and the structure of the copolymers [34]. 2 (-4-Hydroxyphenylazo-) benzoic acid has been used as the MALDI-matrix. The statistical model of mass analysis of copolymers (MACO) has been applied to perform calculations leading to the solution of the complex MALDITOF-MS spectrum. The task was difficult since the difference between successive units was 1 g/mol. The studies were conducted for peaks within the region of 1000e3000 m/z [34]. The MACO calculation program is extremely useful because it eliminates the problem of multiple peaks assignment. The calculations in the MACO program are carried out for a large number of combinations taking into account the sequence of the copolymer chain and the groups found at each end of the chain. The resolution of the spectra obtained using the MALDI-TOF-MS technique depends on the method of data reception and recording. In the case of copolymers, more complicated MALDI-MS spectra are obtained compared to homopolymers. Taking into account the collection of data during the measurement and their subsequent analysis, the spectra of copolymers are more demanding than in the case of spectra of other homogeneous polymers. The method of collecting data in the MALDI-TOF-MS method is very important for the quality of the obtained spectra. MALDI-TOF-MS gives more accurate results in determining the groups present at each end of the copolymer compared to other techniques for characterizing polymers such as nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). In our opinion, additional research is needed to test the MACO program on oligomers within the region of 300e1000 m/z, because in this case MACO will be more universal for testing.
medium of reaction. The process occurs in the presence of zinc(II) chloride or sulfuric acid acting as the catalyst. Currently, the synthetic polymeric materials containing built-in cellulose acetate into the main chain is the aim of the studies [35]. For this purpose, the 1,2-intermolecular radical addition method (1,2-IRA) is used. Moreira et al. synthesized the copolymers containing cellulose acetate and styrene [35]. The characterization of the properties of the obtained polymers is carried out using the MALDI-MS method. The materials containing cellulose acetate are used for the production of various elements that adversely affect the environment, e.g. cigarette filters [35]. Thus, the studies on enzymes that degrade modified cellulose are very popular [36]. LPMOs cause the cellulose acetate degradation in the same way as the degradation of cellulose [16,36]. In the case of a polymer chain containing cellulose acetate, the degradation takes place on the backbone of the chain and on the acetyl group of the polymer [36]. 4.2. Modified starch Ren et al. obtained the modified starch using 1,4-a-glucan branching enzyme (GBE) and tapioca starch [37]. The starch from tapioca has resistant starch which reduces the risk of diseases resulting from an incorrect diet. For this reason, the investigations
Table 1 Collection the main recent developments of MALDI-MS method including the type of polymer sample. The type of polymer sample
The main recent developments of MALDI-MS method
biopolymer
the laser pulses with a frequency of 50 Hz through the use of nitrogen lasers and by this the measurement time of a sample is less than 30 s [11] the measurement time of a sample is less than 30 s [11] the combination of the MALDI-TOF-MS method with the VITEK® 2 test is used in the diagnosis of microorganisms [12] the analysis of the thermolabile molecules without fragmentation molecular ions [21] the precise determination of sequences in the copolymer chain [32] the use of the MACO program for the polymer chain analysis [34] the characterization of a highly branched structure of modified starch [38] the precise determination of the sites of chain degradation of polymers containing cellulose acetate [36]
synthetic polymer
4. Modified polymers 4.1. Modified cellulose Cellulose acetate belongs to the group of modified polymers. Cellulose acetate is obtained as the result of the acetylation reaction of cellulose with acetic anhydride in glacial acetic acid as the
modified polymer
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Table 2 Comparison of MS techniques for analysis of polymers. Method
Major advantages
Major disadvantages
Electrospray ionization - Mass Spectrometry (ESI - MS)
handling samples with large masses [41] the ability to analyze biological samples in which non-covalent interactions occur [41]
Liquid Secondary Ion Mass Spectrometry (LSIMS)
no gas load during measurements (lower noise) [42] easy spraying of high weight ions [42] high sensitivity [43] multidimensional method of studies [43] automation [43] low sample volumes [43] low cost [43] automation [43] high resolution [43] sensitivity from millimolar to nanomolar [43] separation of isobaric cooligomers [43] studies of thermally labile compounds [43]
the results obtained for the mixtures are unreliable [41] many charges occurring with molecular ions result in a lack of data transparency [41] occurrence of mass interferences [42] dependence of secondary ions on the matrix [42] restricted mass range of studied samples [43] sensitive to interfering compounds [43]
Liquid chromatography - Mass Spectrometry (LC - MS) Capillary electrophoresisemass spectrometry (CE - MS) Gas Chromatography - Mass Spectrometry (GC - MS) High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC/ ESI-MS) Inductively Coupled Plasma - Mass Spectrometry (ICP - MS)
excellent detection limits [44] powerful semiquantitative analysis [44] wide dynamic range [44]
on starch as well as modified starch are very important. MALDITOF-MS is a useful method to determine the molecular mass distribution of the modified starch. 2,5-Dihydroxybenzoic acid is used as the matrix in these studies. The results of the studies show that the peaks corresponding with m/z can be equal to the molecular weight because the molecule has the one positive charge. Moreover, modified starch is also obtained by the use of a thermostable branched enzyme from Rhodothermus obamensis and amylomaltase from Thermus thermophiles [38]. The aim of the studies was to obtain modified starch with a highly branched structure. MALDITOF-MS has been used to investigate the molecular mass distribution of the obtained products of modified starch. The spectra contained peaks above the value 4000 m/z [38]. Moreover, in these studies the formation of small cyclodextrins was examined by MALDI-TOF-MS. The smallest identified cyclodextrin was the gcyclodextrin present in the spectra at 1320 m/z. The low values of Molecular weight [Mw] allow formation of cyclostructures during the reaction, such as inter alia cyclo-amylose and the clusters of cyclo-amylopectin [38]. In the case of studies on modified polymers, it should be kept in mind that the amount of modified polymer sample needed to carry out the MALDI-MS study is the relative content of the polymer in the test sample, and it is not the absolute amount of modified polymer in the specified solvent. Obtaining a calibration curve using identical samples of modified polymers is impossible due to the heterogeneity and polydispersity of polymers. In order to determine the content of precursor polymer in the sample of the obtained modified polymer, the method combining the use of an internal standard and the addition of a standard substance is used. Future research on MALDI-MS development should focus on solving the problem of signal attenuation in complex mixtures, e. g. containing samples of various modified polymers mainly modified cellulose and modified starch. On the other hand, it is also possible to refine the method of decomposition of modified starch so that complicated mixtures of the cyclodextrins are not formed, and the problem of signal attenuation could be avoided. 5. Data imaging and data processing in MALDI MALDI imaging is subject to continuous improvement, thanks to the new techniques currently used, such as 3D MALDI-imaging,
not suited for protein characterized by a weight larger than 20 kD [43]
slow analysis time [43] limited number of analyzed molecules [43] sensitive to salts [43] studies on only polar sample [43] complexity of method [43]
argide and matrix interferences, polyatomic interference [44] plasma tail in light path [44]
high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR) and MALDI-Orbitrap-imaging [39]. MALDIFTICR and 3D MALDI-imaging are often used to investigations of biopolymers and metabolites. 3D MALDI-imaging is the method without marking [39]. It is recommended to use magnetic resonance imaging in combination with 3D MALDI-imaging in order to improve the interpretation of MALDI images [39]. Modern research proves that MALDI-Orbitrap-imaging allow to a quick and easy analysis. Moreover, the advantage of this method is a miniature design. Data processing in MALDI-MS consists of two stages - first, preprocessing and secondly statistical analysis [40]. The spectra are subjected to the following rotation: the removal of the background, normalization of intensity, and alignment [40]. The simpler the preprocessing method, the further processing of data is faster. The availability and diversity of data for analysis using the MALDI-MS method is constantly increasing, but there is a problem with the reduction of measurement errors. 6. Conclusions This review shows that the MALDI-MS technique is a powerful analytical tool to study and characterize not only synthetic polymers but also polymers obtained from natural sources. The solvent is not required in the MALDI-MS method; therefore optimizations of this method can eliminate this dependence. In contrast, the image of spectra obtained by this method is dependent on the matrix and the method of preparing the sample for measurements. The main recent developments of MALDI-MS method for studies of polymers have been summarized in Table 1. Moreover, the comparison of MALDI-MS with other MS techniques is presented in Table 2. The future prospects in field of MALDI-MS method to different types of polymeric materials have been described in detail. Acquiring new polymeric materials is the subject of contemporary research [45e47]. There are ongoing studies on new catalysts of polymerization reactions, in particular on non-metallocene complex compounds containing Cr3þ and V4þ cations [48e57]. The results of the studies on the catalytic activity of these complex compounds are promising. The synthesis of new polymeric
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materials consequently brings with it the development of the MALDI-MS technique for the characterization and analysis of polymers [34,58]. We predict that in the near future it will be possible to further explore the structure of polymers and the obtained MALDI-MS spectra will be even higher resolution than those present due to the innovative approach to sample analysis inter alia through computer techniques and simulations associated with it. Acknowledgements This work was supported by the National Science Centre, Poland under Grant number 2015/19/N/ST5/00276. References [1] M. Karas, D. Bachmann, F. Hillenkamp, Influence of the wavelength in highirradiance ultraviolet laser desorption mass spectrometry of organic molecules, Anal. Chem. 57 (1985) 2935e2939. https://doi.org/10.1021/ac00291a042. [2] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, T. Matsuo, Protein and polymer analyses up to m/z 100 000 by laser ionization timeofflight mass spectrometry, Rapid Commun. Mass Spectrom. 2 (1988) 151e153. https://doi. org/10.1002/rcm.1290020802. [3] M. Karas, U. Bahr, Laser desorption ionization mass spectrometry of large biomolecules, Trac. Trends Anal. Chem. 9 (1990) 321e325. https://doi.org/10. 1016/0165-9936(90)85065-F. [4] F. Hillenkamp, J. Peter-Katalinic (Editors), MALDI MS: a Practical Guide to Instrumentation, Methods and Applications, second ed., John Wiley & Sons, Hoboken (New Jersey), 2013. [5] W. Pusch, M. Kostrzewa, Application of MALDI-TOF mass spectrometry in screening and diagnostic research, Curr. Pharmaceut. Des. 11 (2005) 2577e2591. https://doi.org/10.2174/1381612054546932. [6] T.R. Sandrin, J.E. Goldstein, S. Schumaker, MALDI TOF MS profiling of bacteria at the strain level: a review, Mass Spectrom. Rev. 32 (2013) 188e217. https:// doi.org/10.1002/mas.21359. [7] G. Montaudo, F. Samperi, M.S. Montaudo, Characterization of synthetic polymers by MALDI-MS, Prog. Polym. Sci. 31 (2006) 277e357. https://doi.org/10. 1016/j.progpolymsci.2005.12.001. [8] A.J. Hoteling, W.J. Erb, R. Tyson, K.G. Owens, Exploring the importance of the relative solubility of matrix and analyte in MALDI sample preparation using HPLC, Anal. Chem. 76 (2004) 5157e5164. https://doi.org/10.1021/ac049566m. [9] R. Skelton, F. Dubois, R. Zenobi, A MALDI sample preparation method suitable for insoluble polymers, Anal. Chem. 72 (2000) 1707e1710. https://doi.org/10. 1021/ac991181u. [10] S. Angeletti, Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology, J. Microbiol. Methods 138 (2017) 20e29. https://doi.org/10.1016/j.mimet.2016.09.003. [11] P. Seng, M. Drancourt, F. Gouriet, B. La Scola, P.E. Fournier, J.M. Rolain, D. Raoult, Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Clin. Infect. Dis. 49 (2009) 543e551. https://doi.org/10.1086/ 600885. [12] S.J. Jo, K.G. Park, K. Han, D.J. Park, Y.J. Park, Direct identification and antimicrobial susceptibility testing of bacteria from positive blood culture bottles by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Vitek 2 system, Ann. Lab. Med. 36 (2016) 117e123. https://doi.org/10. 3343/alm.2016.36.2.117. [13] B. Karolski, L.O. Cardoso, L.H. Gracioso, C.A. Nascimento, E.A. Perpetuo, MALDIBiotyper as a tool to identify polymer producer bacteria, J. Microbiol. Methods 153 (2018) 127e132. https://doi.org/10.1016/j.mimet.2018.09.016. [14] M.W. Linscheid, Molecules and elements for quantitative bioanalysis: the allure of using electrospray, MALDI, and ICP mass spectrometry sidebyside, Mass Spectrom. Rev. 38 (2019) 169e186. https://doi.org/10.1002/mas.21567. [15] Y. Fujii, J. Xu, T. Fujino, Chirality and structure-selective MALDI using mold matrix, Sci. Rep. 8 (2018) 13138. https://doi.org/10.1038/s41598-018-31629-6. [16] B. Westereng, J.S. Loose, G. Vaaje-Kolstad, F.L. Aachmann, M. Sørlie, V.G. Eijsink, Analytical tools for characterizing cellulose-active lytic polysaccharide monooxygenases (LPMOs), in: M. Lübeck (Editor), Cellulases. Methods in Molecular Biology, Humana Press, New York, 2018, pp. 219e246. https://doi.org/10.1007/978-1-4939-7877-9_16. [17] I.J. Kim, N. Seo, H.J. An, J.H. Kim, P.V. Harris, K.H. Kim, Type-dependent action modes of Tt AA9E and Ta AA9A acting on cellulose and differently pretreated lignocellulosic substrates, Biotechnol. Biofuels 10 (2017) 46. https://doi.org/ 10.1186/s13068-017-0721-4. [18] D. Wang, Y. Sun, L. Chang, Z. Tong, Q. Xie, X. Jin, L. Zhu, P. He, H. Li, X. Wang, Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis, J. Proteomics 182 (2018) 53e64. https://doi.org/10.1016/j.jprot. 2018.05.002.
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