Tertiary Matrices for the Analysis of Polyethylene Glycols Using MALDITOF MS
 DOI : 10.5478/MSL.2014.5.2.49
 Author: Hong Jangmi, Kim Taehee, Kim Jeongkwon
 Organization: Hong Jangmi; Kim Taehee; Kim Jeongkwon
 Publish: Mass Spectrometry Letters Volume 5, Issue2, p49~51, 30 June 2014

ABSTRACT
The effectiveness of tertiary matrices composed of the combination of three common matrices (dihydrobenzoic acid (DHB), αcyano4hydroxycinnamic acid (CHCA), and sinapinic acid (SA)) was compared with that of single or binary matrices in the analysis of polyethylene glycol (PEG) polymers ranging from 1400 to 10000 Da using matrixassisted laser desorption/ionization timeofflight mass spectrometry (MALDITOF MS). A tertiary matrix of 2,5DHB+CHCA+SA was the most effective in terms of S/N ratios. CHCA and CHCA+SA produced the highest S/N ratios among the single matrices and the binary matrices, respectively. The improvement observed when using a tertiary matrix in analyses of PEG polymers by MALDITOF MS is believed to be due to the uniform morphology of the MALDI sample spots and synergistic effects arising from the mixture of the three matrix materials.

KEYWORD
MALDITOF MS , tertiary matrix , polyethylene glycols , 2,5dihydrobenzoic acid , αcyano4hydroxycinnamic acid , sinapinic acid

Introduction
Matrixassisted laser desorption/ionization timeofflight mass spectrometry (MALDITOF MS) is generally used in analyses of samples such as proteins, peptides, polyethylene glycol (PEG) polymers, and carbohydrates. Different types of analyte molecules often require different matrices for optimal results. Common matrices are 2,5dihydrobenzoic acid (2,5DHB), αcyano4hydroxycinnamic acid (CHCA), and sinapinic acid (SA). A binary matrix of two such matrix materials is reportedly superior to a single matrix due to improved homogeneity of the sample surface.^{1,2}
Due to its unique properties, including nontoxicity, nonimmunogenicity, hydrophilicity, and high flexibility, PEG is commonly used in drug delivery strategies,^{3} nanoparticle coatings,^{4} and crosslinkers.^{5} PEG polymers have been analyzed via MALDITOF MS with matrices of 2,5DHB,^{6,7} 2,6DHB,^{8} norharmane,^{9} or dithranol.^{10}
In this study, three different matrix materials were combined to form a tertiary matrix that was then applied to MALDITOF MS analyses of PEG polymers with number average molecular weights (Mn) ranging from 400 to 10000. The effectiveness of these tertiary matrices was compared with those of single and binary matrices of the same materials.
Experimental
PEG (average Mn ~400), PEG (average Mn ~1450), PEG (average Mn ~3350), PEG (average Mn ~8000), PEG (average Mn ~10000), 2,5dihydroxybenzoic acid (2,5DHB), 2,6dihydroxybenzoic acid (2,6DHB), αcyano4hydroxycinnamic acid (CHCA), sinapinic acid (SA), trifluoroacetic acid (TFA), acetonitrile (ACN), and sodium trifluoroacetate (NaTFA) were purchased from SigmaAldrich (St. Louis, MO, USA). The MALDI plate was obtained from Hudson Surface Technology, Inc. (Newark, NJ, USA). All mass spectra were acquired using an Axima CFR MALDITOFMS (Shimadzu Biotech) equipped with a 337nm N_{2} laser in positive reflectron mode for PEG1450 and PEG3350 or in positive linear ion mode for PEG8000 and PEG10000. The S/N ratios were obtained using the Shimadzu Biotech Launchpad software (v.2.9.2).
To prepare single matrices, each of 10 mg DHB, CHCA, or SA was dissolved in 1 mL of 50% ACN/0.1% TFA in distilled water. To prepare binary matrices, each of 10 mg matrix material (a total of 20 mg matrix) was dissolved in 1 mL of 50% ACN/0.1% TFA in distilled water. The tertiary matrices were prepared by dissolving each of 10 mg matrix (a total of 30 mg matrix) in 1 mL of 50% ACN/0.1% TFA in distilled water. PEG samples (1.0 mM) (Mn 1450, 3350, 8800, 10000, and 20000) were prepared by dissolving 1 μmol PEG in 1.0 mL of 0.5% TFA in water.
Each PEG sample was mixed with each matrix at an equal volume ratio. To load the sample, 1 μL of the mixture was loaded onto a stainless steel plate. After the spots were completely dried, 1 μL of 10 mM NaTFA was loaded on top of the spots. The amount of PEG polymer loaded onto each sample spot was 500 pmol.
Results and discussion
Two tertiary matrices, 2,5DHB+CHCA+SA and 2,6DHB+CHCA+SA, were prepared along with four single matrices, 2,5DHB, 2,6DHB, CHCA, and SA, and five binary matrices, 2,5DHB+CHCA, 2,5DHB+SA, 2,6DHB+CHCA, 2,6DHB+SA, and CHCA+SA. Four different PEG polymers (PEG1450, PEG3350, PEG8000, and PEG10000) were analyzed with each matrix.
Table 1 presents the S/N ratios obtained for the various PEG polymers in MALDITOF MS analyses using the 11 matrices described above. Figure 1 shows the MALDI mass spectra obtained with representative matrices in each class: CHCA, 2,5DHB+CHCA, and 2,5DHB+CHCA+SA. The most effective matrix among all of the investigated matrices was the tertiary matrix 2,5DHB+CHCA+SA. The most effective single matrix was CHCA, followed by SA. The addition of CHCA to any matrix significantly increased S/N ratios. Binary matrices containing CHCA yielded higher S/N ratios than their counterpart binary matrices without CHCA.
The 2,6DHB matrix provided lower S/N values compared to the 2,5DHB matrix, which is different from our previous investigation,^{8} when 2,6DHB provided better detection for the MALDITOF MS analysis of PEG polymers. The sample preparation methods presumably affect the effectiveness of the detection of PEG polymers, whereby 2,6DHB is effective with a threelayer preparation method as shown in our previous study. The importance of MALDI sample preparation method was already demonstrated whereby a different optimum sample preparation method was observed for each protein digest.^{11}
Figure 2 shows the images of the MALDI sample spots for the 11 different matrices with PEG1450. Very similar patterns were observed from the sample spots of single matrices of CHCA and SA, in which scattered small white aggregations were observed. More uniform small aggregations were observed in the binary matrix of CHCA+SA, whose spot provided the most uniform pattern among the binary matrices. The spots of the two tertiary matrices also showed a uniform pattern, which is believed to be from the contribution of CHCA and SA. Here, we believe this uniform morphology results in homogeneous sample distribution and contributes to the increased sensitivity. In addition, the observation that slightly improved performance from the tertiary matrices (DHB+CHCA+SA) compared to the binary matrix of CHCA+SA could be explained by the contribution of DHB matrices, resulting in a synergic effect.
Conclusion
Mixtures of three different matrices, which are 2,5DHB+CHCA+SA or 2,6DHB+CHCA+SA, provided an improved sensitivity in the analysis of PEG polymers using MALDITOF MS. The improvement is believed to be due to the uniform morphology by mixing CHCA and SA matrices, along with the unique contribution from DHB matrices.

1. Shanta S. R., Zhou L. H., Park Y. S., Kim Y. H., Kim Y., Kim K. P. 2011 [Anal. Chem.] Vol.83 P.1252

2. Yang C., Hu X., Loboda A. V., Lipson R. H. 2010 [J. Am. Soc. Mass Spectrom.] Vol.21 P.294

3. Knop K., Hoogenboom R., Fischer D., Schubert U. S. 2010 [Angew. Chem. Int. Ed.] Vol.49 P.6288

4. Junejo Y., Baykal A., Sozeri H. 2013 [Eur. J. Chem.] Vol.11 P.1527

5. Scott R. A., Peppas N. A. 1999 [Biomaterials] Vol.20 P.1371

6. de Koster C. G., Duursma M. C., van Rooij G. J., Heeren R. M., Boon J. J. 1995 [Rapid Commun. Mass Spectrom.] Vol.9 P.957

7. Hanton S. D., Cornelio Clark P. A., Owens K. G. 1999 [J. Am. Soc. Mass Spectrom.] Vol.10 P.104

8. Lee A., Yang H. J., Kim Y., Kim J. 2009 [Bull. Korean Chem. Soc.] Vol.30 P.1127

9. ErraBalsells R., Nonami H. 2003 [Arkivoc] Vol.2003 P.517

10. Enjalbal C., Ribiere P., Lamaty F., YadavBhatnagar N., Martinez J., Aubagnac J. L. 2005 [J. Am. Soc. Mass Spectrom.] Vol.16 P.670

11. Padliya N. D., Wood T. D. 2008 [Anal. Chim. Acta] Vol.627 P.162

[Table 1.] Summary of S/N ratios for PEG polymers in different matrices

[Figure 1.] MALDI mass spectra of PEG1450, PEG3350, PEG8000, and PEG10000 in the matrices of (AD) CHCA, (EH) binary matrix (2,5DHB+CHCA), and (IL) tertiary matrix (2,5DHB+CHCA+SA). The yaxis shows the relative intensity. Mainly [PEG+Na]+ ions were observed, where PEG indicates HO(CH2CH2O)nH.

[Figure 2.] MALDI sample spot images of 11 different matrices for the analysis of PEG1450.