Analytica Chimica Acta 561 (2006) 16–24 Investigation of the enzyme Bacillus agaradhaerens Cel 5a as an analytical tool in mass spectral characterisation of methylcelluloses Arieh S. Cohen a,∗ , Carina Nilsson b , Herje Schagerl ¨ of c , Folke Tjerneld c , Lo Gorton b a Department of Occupational and Environmental Medicine, Lund University Hospital, SE-221 85, Lund, Sweden b Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-22 100 Lund, Sweden c Department of Biochemistry, Lund University, P.O. Box 124, SE-22 100 Lund, Sweden Received 7 December 2005; received in revised form 9 January 2006; accepted 9 January 2006 Abstract Two methylcelluloses (MC) were investigated by partial hydrolysis followed by direct infusion electrospray ionisation mass spectrometry (ESI- MS). Partial hydrolysis was achieved either enzymatically or by acid hydrolysis. Enzyme hydrolysis was performed using the endoglucanase Bacillus agaradhaerens Cel 5a. Three different hydrolysis buffer solutions were investigated in order to determine which was most compatible with the subsequent MS analysis. ESI-MS experiments showed that when using enzymatic hydrolysis as an analytical tool for characterisation of modified cellulose one has to be watchful for the investigated polymer to have a sufficient amount of available cleavage sites in order to ensure that the original ends of the polymer do not influence the results. MS 2 experiments confirmed that the detected products originated from methylcellulose and provided structural information on the investigated products. MS data proved that B. agaradhaerens Cel 5a is able to cleave 1 → 4 glycosidic linkages adjacent to a permethylated glucosyl unit. MS 2 data showed that methylation on the C-2 and C-6 hydroxyl groups of the glucosyl unit on the non-reducing side of the cleavage site hindered the enzyme. © 2006 Elsevier B.V. All rights reserved. Keywords: Methylcellulose; MC; Mass spectrometry; ESI-MS; Bacillus agaradhaerens Cel 5a; Cellulase; Modification pattern 1. Introduction Methylcellulose (MC) is a soluble cellulose derivative. It is finding increasing industrial use (e.g. in pharmaceuticals, paints, etc.) as MC is made from a readily renewable resource. Previ- ous investigations of MC and other cellulose derivatives show that the degree of substitution as well as the substituent distribu- tion affect the physical and chemical properties of the modified cellulose [1–7]. In order to determine the substituent distribu- tion several strategies have been applied, e.g. NMR analysis, acid hydrolysis, enzyme hydrolysis, GC-MS, LC-MS, SEC- multi angle light scattering (MALS), often in combination [2]. Enzyme hydrolysis followed by analysis of the liberated prod- ucts utilizes the fact that the modification hinders the enzyme from functioning properly, yielding a selective partial hydroly- sis. Using knowledge of enzyme selectivity, it is possible to use ∗ Corresponding author. Present address: Department of Occupational and Environmental Medicine, Lund University, Lund University Hospital, SE-22 185 Lund, Sweden. Tel.: +46 46 17 31 98; fax: +46 46 14 37 02. E-mail address: arieh.cohen@ymed.lu.se (A.S. Cohen). the liberated products to elucidate structural information on the investigated MC. Conversely, product investigation along with knowledge of the polymer allows for investigation of enzyme selectivity. Mass spectral analysis has been used for the investigation of hydrolysed MC (and other cellulose polymers). Previously, MALDI-TOF has been used for both the analysis of MC and car- boxymethyl cellulose [8,9]. MALDI-TOF is able to scan much higher in m/z than ESI-MS and thus is better suited for the anal- ysis of long chain polymers. ESI-MS is able to scan below 500 m/z, a mass range, where most MALDI-TOF instruments are inefficient. Thus, ESI-MS is appropriate for the investigation of short chain degradation products [2,3,10]. ESI-MS also offers the possibility to perform MS 2 experiments, which offer struc- tural information. In this study, partial hydrolysis was achieved with the enzyme Bacillus agaradhaerens Cel 5a (BaCel 5a) and by partial acid hydrolysis with trifluoroacetic acid (TFA). The hydrolysis prod- ucts were analysed with direct infusion electrospray ionisation mass spectrometry (ESI-MS) and all of the detected hydroly- sis products were investigated with tandem mass spectrometry 0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.01.019