Published: September 29, 2011 r2011 American Chemical Society 8468 dx.doi.org/10.1021/ac2017103 | Anal. Chem. 2011, 83, 8468–8476 ARTICLE pubs.acs.org/ac Differentiation of Closely Related Isomers: Application of Data Mining Techniques in Conjunction with Variable Wavelength Infrared Multiple Photon Dissociation Mass Spectrometry for Identification of Glucose-Containing Disaccharide Ions Sarah E. Stefan, † Mohammad Ehsan, † Wright L. Pearson, †,|| Alexander Aksenov, †,^ Vladimir Boginski, ‡ Brad Bendiak, § and John R. Eyler †, * † Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611-7200, United States ‡ Department of Industrial & Systems Engineering, University of Florida, 1350 North Poquito Road, Shalimar, Florida 32579-1163, United States § Department of Cellular and Developmental Biology and Program in Structural Biology and Biophysics, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045, United States b S Supporting Information I somers remain a problem in mass spectrometry, especially closely related isomers that yield very similar ratios of product ions upon unimolecular dissociation. This situation is frequently encountered among isomers varying in their stereochemistry at one or more positions (e.g., carbohydrates and glycoconjugates 1À3 ), positional isomers (e.g., phospholipids, di- and triacylglycerols 4À6 ), and isomers varying in the nature of double bonds (cis or trans or multiple double bonds, as in various lipids, wax esters, hydro- carbons, and other compounds 7À9 ). The problem has been approached in different ways, some more successful than others, depending on the nature of the precursor ions. Use of differences in fragmentation patterns after collision-induced dissociation (CID) is still the most common and well-studied approach. 1À3,5À7,9,10 Other techniques exploit differences in physical properties of isomeric ions: migration rates of ions through a neutral gas (ion mobility spectrometry), 11,12 reactivities of ions, 13,14 charge exchange MS, 15,16 direct photon absorption or emission, 17,18 variable wavelength photodissocia- tion in the infrared, 19À23 visible 24 or ultraviolet 25 with one or more 26,27 lasers, differences in photodissociation by rapid pulse shaping over an approximate Gaussian wavelength distribution centered at 800 nm, 28,29 and relatively high-resolution spectroscopy in cold traps. 30,31 In cases relying on dissociation of a precursor ion, the mass spectra may be too similar to assign particular isomers from the product ion ratios with confidence. This can occur for several reasons: (1) product ion ratios frequently differ somewhat among different instruments; (2) product ion ratios can vary somewhat using the same instrument under supposedly identical conditions; (3) methods of processing digitized spectral data vary, and at least some error is introduced in quantitation of spectral peaks depending on the processing method; (4) white noise is present to varying extents; and (5) a proper statistical evaluation of data is often not performed. Data mining is a set of techniques for evaluation and grouping of complex sets of data depending on multiple selected criteria. 32,33 In mass spectrometry, the analyses can be applied to virtually any quantitative values (e.g., product ion intensities at different m/z values, multiple secondary or tertiary product ion intensities in MS 2 or MS 3 experiments, chromatographic retention times, ion mobility drift times) which exhibit a difference among the target Received: July 2, 2011 Accepted: September 29, 2011 ABSTRACT: Data mining algorithms have been used to analyze the infrared multiple photon dissociation (IRMPD) patterns of gas-phase lithiated disaccharide isomers irradiated with either a line-tunable CO 2 laser or a free electron laser (FEL). The IR fragmentation patterns over the wavelength range of 9.2À10.6 μm have been shown in earlier work to correlate uniquely with the asymmetry at the anomeric carbon in each disaccha- ride. Application of data mining approaches for data analysis allowed unambiguous determination of the anomeric carbon configurations for each disaccharide isomer pair using fragmen- tation data at a single wavelength. In addition, the linkage positions were easily assigned. This combination of wavelength-selective IRMPD and data mining offers a powerful and convenient tool for differentiation of structurally closely related isomers, including those of gas-phase carbohydrate complexes.