ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2011–2012 David J. Harvey* Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford OX1 3QU, UK Received 19 June 2014; accepted 15 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21471 This review is the seventh update of the original article published text. As the review is designed to complement the earlier work, in 1999 on the application of MALDI mass spectrometry to the structural formulae, etc. that were presented earlier are not analysis of carbohydrates and glycoconjugates and brings repeated. However, a citation to the structure in the earlier work coverage of the literature to the end of 2012. General aspects is indicated by its number with a prefix (i.e., 1/x refers to such as theory of the MALDI process, matrices, derivatization, structure x in the first review and 2/x to structure x the second). MALDI imaging, and fragmentation are covered in the first part Books, general reviews, and review-type articles directly of the review and applications to various structural types concerned with, or including, MALDI analysis of carbohydrates constitute the remainder. The main groups of compound are and glycoconjugates to have been published during the review oligo- and poly-saccharides, glycoproteins, glycolipids, glyco- period are listed in Table 1. Reviews relating to more specific sides, and biopharmaceuticals. Much of this material is presented aspects of MALDI analysis are listed in the appropriate sections. in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions, and II. THEORY applications to chemical synthesis. # 2015 Wiley Periodicals, Inc. Mass Spec Rev Investigations into the mechanisms operating during MALDI Keywords: MALDI; carbohydrates; glycoproteins; glycolipids; ionization continue. Knochenmuss and Zhigilei (2012) have biopharmaceuticals pointed out that, although ion recombination is an important process in determining the number of ions observed, this reaction has received comparatively little attention. In their I. INTRODUCTION study, molecular dynamics simulations were used to investigate the process and a tunneling model was found to explain some of This review is a continuation of the seven earlier ones in this the observations. They propose that “tunneling recombination in series on the application of MALDI mass spectrometry to the the Marcus inverted region may be a major reason why MALDI analysis of carbohydrates and glycoconjugates (Harvey, 1999, is a viable analytical method by allowing ion formation to 2006, 2008, 2009, 2011a, 2012, 2015) and is intended to bring exceed ion loss on the time scale of the plume expansion.” the coverage of the literature to the end of 2012. It is hoped that Jaskolla and Karas (2011) have devised an experiment to this review is relatively comprehensive but this is becoming show that both of the postulated mechanisms for ion production increasingly difficult in view of the massively increasing number by protonation under MALDI conditions, the Lucky Survivor of papers and journals published each year and the splitting of and the gas-phase protonation models, exist together. The papers between the main text and “supplementary material.” experiment used the deuterated t-butyl ester of a-cyano-4- Some papers published in the review period will invariably have hydroxycinnamic acid (CHCA, 1/23) as the matrix. This been overlooked if the technique is not deemed sufficiently compound is stable under typical sample preparation conditions important to appear in the main text of an article. An excessive but generates deuterated reagent ions, including the deuterated use of acronyms and abbreviations, is another problem with and deuterium-free matrix acid, only upon laser irradiation. The modern scientific writing. A recent Editorial in Nature Methods generation of deuterated analyte ions proves the existence of the (Editorial, 2011) cautions against the excessive or inappropriate gas-phase protonation model and the detection of protonated use of acronyms, particularly for new experimental techniques analytes by application of deuterated matrix compounds without but cites MALDI as an example of the good and appropriate use acidic hydrogens proves the survival of analytes pre-charged of an acronym. from solution. The observed ratio of ions from the two ionization MALDI continues to be a major technique for the analysis processes depends on the applied experimental parameters as of carbohydrates; Figure 1 shows the year-by-year increase in well as the nature of the analyte and matrix. Increased laser papers reporting use of the technique for the period 1987–2012. fluences and lower matrix proton affinities favor gas-phase Slightly fewer papers than expected were found but this may protonation, whereas more protonation in solution leads to more relate, as mentioned above, to absence of MALDI in the main Lucky Survivors. Another feature of a MALDI ion source is the angle at ÃCorrespondence to: David J. Harvey, Department of Biochemistry, which the sample/matrix plume is emitted. To address this issue, Oxford Glycobiology Institute, University of Oxford, Oxford OX1 Liang et al. (2011b) have measured the high-resolution angular 3QU, UK. E-mail: [email protected] and velocity distributions for the neutral analytes, tryptophan, Mass Spectrometry Reviews, 2015, 9999, 1–168 # 2015 by Wiley Periodicals, Inc. & HARVEY FIGURE 1. Numbers of papers per year relating to the application of MALDI MS to the study of carbohydrates and glycoconjugates. and poly-tryptophan ionized with 2,4,6-trihydroxyacetophenone beginning of desorption when the surface temperature was high. (THAP, 1/44) using 355 nm laser desorption. The results This expansion resulted in a large velocity normal to the surface suggested that two separate mechanisms dominate the angular and a very narrow angular distribution. Before the end of and velocity distributions at the beginning and before the end of desorption, the surface temperature decreased and thermal desorption. A molecular jet-like isentropic (constant entropy) desorption at low vapor pressure was the dominant process. expansion was found to dominate the plume expansion at the Under these conditions, the velocities become small and the TABLE 1. General reviews No. of Subject Notes Reference citations In situ pressure probe sampling and UV-MALDI (Gholipour et Plant cells 37 MS for profiling metabolites in living single cells al., 2012) Several techniques. MALDI (Ho & Reddy, Identification of pathogens 260 detection of lipid A etc. 2011) (Horn & Lipidomics in tissues, cells and subcellular General review, some 102 Chapman, compartments glycolipids 2012) High-resolution plant metabolomics: whole-cell (Kueger et al., Short general review 139 analysis to subcellular metabolite distributions 2012) (Oikawa & Metabolite analyses of single cells Small section on MALDI 88 Saito, 2012) Gold nanomaterials as a new tool for bioanalytical Applications to several (Pilolli et al., applications of laser desorption ionization mass 97 structural types 2012) spectrometry Short general review on characterization of mainly (Robinson et GAGs N-links, glycan arrays 224 acidic glycans al., 2012) Application of surface plasmon resonance for the Section on MALDI and in 201 (Safina, 2012) detection of carbohydrates and glycoconjugates combination with SPR A review of the progress in (Schiller et al., TLC/HPTLC with mass spectrometric detection 98 the last 5 years 2011) Thin-layer chromatography and mass spectrometry Several compound types - (Tuzimski, for the separation and detection of large and small 228 carbohydrates peptides etc. 2011) biomolecules Review of reactions (Urban et al., -on-a- performed on MALDI plates. 332 2011) Tables of applications Mainly N- and O-linked glycans. Most rearrangements (Wuhrer et al., Glycan rearrangements 51 associated with presence of a 2011) proton 2 Mass Spectrometry Reviews DOI 10.1002/mas ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & angular distribution increases. Unlike the initial situation where in ambient MALDI plumes. A large number of nanoparticles much analyte is present in the plume, only a very small amounts were produced, with average particle diameters ranged from 40 of analyte are generated by thermal desorption. to 170 nm depending on the matrix and the laser fluence. CHCA The relationships between MALDI ion yield(s) as a and sinapinic acid also produced particles in the range of 10 to function of desorption alone and (or) ionization have been 30 nm, which was attributed to the direct ejection of clusters. investigated using two model systems. In the first model system, The work has relevance to ionization methods such as matrix- b-cyclodextrin (b-CD, 4/6), which could be directly desorbed, assisted laser desorption electrospray ionization (MALDESI) was analyzed with and without a silicon-based heat sink where it will be important to produce larger particulates to compound. The sink allowed heat to pass through but obstructed facilitate analyte transfer to the charged electrospray droplet. the flow of charge. In the second model system, substance P, Gas-phase basicities of several MALDI matrices have been which could not be desorbed, was analyzed under similar measured using an electrosonic spray ionization source. Mea- conditions. The ion yields from b-CD under either