30 May / June 2019


matter is presented in Figure 3 I and II, APTS N-glycans from formalin-fixed, paraffin- embedded mouse tissue specimens, were analysed [22] and an attempt at glycome sequencing was realised using different exoglycosidases [23].


Earlier, Callewaert et al. [24] were able to help liver cirrhosis diagnosis by using CE/ LIF in quite the same conditions as the ones described above [25], the log of the ratio of peak 7 and 8 from Figure 4 allowing a diagnostic of the pathology.


Mass spectrometry can be used as detector after the CE/LIF system. To minimise the differences in migration times, the LIF detector can be connected just before the ESI source [26].


b) Amino acids and biogenic acids.


○, β-linked GlcNAc;●, β-linked galactose , □, α-linked mannose; ▪, β-linked mannose; ∆, α-1,6-linked fucose ,


Figure 4: Separations of APTS labelled glycans. Top, maltooligosaccharide reference. Middle, typical electropherogram of desialylated N-glycans derived from proteins in control serum sample. Nine peaks are clearly visible in the full detection range, with five more in the ×10 blow-up of the latter part of the electropherogram. Bottom, representative electropherogram obtained from cirrhosis case. Structures of N-glycans of relevance to this study are shown below the panels; peaks that are important for fibrosis/


cirrhosis markers are boxed [24].


published [12]. For LIF studies pulsed laser can be used. Two articles summarise the photodegradation processes using these high pulsated power lasers. The first mathematically describe the process of the pulsed photodegradation [13 ] and the second shows that photodegradation will drive to get non-linear calibration curves when the range of concentrations is of two decades [14].


III. The main applications.


Many applications are described in the literature; however, the focus will be on the ones considered most important.


a) Glycans.


The review articles of Mantovani et al. [15] and Lu et al. [16] underlined the use of LIF detection for glycan applications, particularly regarding the glycan part of proteins, especially for N-glycosylation. For these molecules, the different steps were greatly optimised. As an example, the first step for N-glycan release from the glycoprotein was run using immobilised PNGase F [17]. The obtained glycans are labelled by reductive amination, with a charged fluorophore


containing a primary amine for example the 8-aminopyrene-1,3,6-trisulfonate (APTS)- which reacts with the aldehyde group at the reducing end of the glycan structures. The Schiff base thus formed is reduced with sodium cyanoborohydride to form a stable conjugate [18]. To minimise the loss of sialic acid, authors have proposed a simple protocol to label the glycans from 100 µg of a glycoprotein in a sample containing THF that evaporates slowly during the derivatisation at 60°C. Using these conditions, the authors demonstrated an increase of sialilated species by a factor two [19]. In another study, carboxyl- coated magnetic microparticles (COOH- beads) were reported to specifically bind polysaccharides and were used for a simple sample preparation for automated analysis. The excess of APTS was removed and the saccharides could be concentrated [20].


Recently, a catalytic hydrogen transfer from formic acid catalysed by water- soluble iridium (III)-phosphine complexes was proposed as an alternative to the cyanoborohydride [21] in an effort to prevent HCN formation.


One of the most impressive results on this


This application has been regularly reviewed since 2001, first by Prata et al. [27], then by Poinsot et al. [28]. Because most of amino acids (AA) are not native fluorescent, a labelling step is necessary. If all the AA and biogenic amines have to be studied, FITC or 3-(4-carboxybenzoyl)quinoline-2- carboxaldehyde (CBQCA) can be used and the derivatives easily separated. Both labels are be excited with a 488nm Argon laser or a LED at 480 nm [29] to get the same sensitivity. The main limitations with FITC are its natural fluorescence and its impurities making the identification of the labelled molecules at low concentrations more difficult, because they could migrate with the impurities. Using MEKC, highly resolved specific separations can be obtained [30] and AA can be identified as taurine [31] (Figure 5 I) or dimethyl arginine [32].


CBQCA is a fluorogenic dye with low levels of impurities, however it cannot label secondary amines e.g. proline (Pro). In addition, Perquis et al. demonstrated that the fluorescent yield of the CBQCA- Trp derivative was 50 times less important than CBQCA-Trp, making Tryptophan (Trp) a difficult AA to be identified [33]. However, very good separations can be obtained using micellar electrokinetic chromatography (MEKC) [34] (Figure 5 II).


A good molecule candidate for better more selection and identification of thiol-containing AA, such as cysteine (Cys) or homocysteine (Hcy) [35], is the iodoacetamido fluorescein. It was used in clinical studies to measure the concentration of Hcy in plasma (Figure 6), to help diagnosis of cardiovascular disease [36,37]. Trp, Tyrosine (Tyr) and their metabolites can be selectively detected using UV pulsed lasers [38].


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