Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins

Abstract

A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M ϩ CH 3 CO 2 ] Ϫ using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M Ϫ H] Ϫ . The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 g of fetuin, ribonuclease B, peroxidase, and ␣ 1 -acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. arbohydrates play vital roles in the control of many key biological processes by acting as reciprocating compounds with proteins in molecular recognition events [1,2]. Associative interactions between oligosaccharides on glycoproteins and lectins on the surfaces of binding partners can be involved in the initiation steps of many diseases such as influenza, cholera, stomach cancer, and cancer metastasis [3]. An understanding of these initiating binding processes at the molecular level requires the structural determination of oligosaccharides and the quantification of the glycan monosaccharide constituents, which would be essential towards the development of possible cures for many diseases [4,5].Monosaccharides compositional analysis is commonly performed using gas-chromatography [6 -10], liquid-chromatography [11-13], or capillary electrophoresis [14 -21]. GC/MS chromatography with an electron-impact ion source has been the traditional method of choice for the quantification of neutral monosaccharides. GC analysis of the trimethylsilyl ether derivatives of sugars is a well-known procedure [9], as is the GC separation of sugars as alditol acetates [7]. However, sample preparation for GC is extensive, involving the derivatization of sugars with specialty reagents to render them more volatile [10]. Also, the sensitive chromatographic operating parameters for GC are not well suited for routine analysis. Liquid chromatography analysis of monosaccharides is complicated by the fact that these compounds do not possess a UV chromophore and consequently, laborious derivatization with chromophores or fluorophores is required before UV or fluorescence detection [22][23][24][25][26]. Neutral sugars have also been analyzed by HPLC using...

Separation of monosaccharides hydrolyzed from glycoproteins without the need for derivatization

“…Briefly, glycoproteins were denatured, and neutral sugars (mannose, galactose, glucose, fructose, and fucose) or amine sugars (GlcNAc and GalNAc) were liberated from their glycoprotein precursors using 2 mol/L trifluoroacetic acid (TFA, Fluka 91699) or 4 mol/L hydrochloric acid (HCl, Fluka 84410), respectively, at 100°C for 4 h. Sialic acids (NANA, NGNA), being terminal subunits and less chemically stable, were hydrolyzed under milder conditions (0.1 mol/L TFA for 2 h at 80°C). N-acetyl groups that were observed to degrade due to the harshness of the hydrolysis conditions, as reported previously [16] for amino sugars, required a re-acetylation step. Re-acetylation was performed for one hour at room temperature by the addition of 50 μL of NH 4 HCO 3 buffer, pH 7.8, with 25 μL of neat acetic anhydride to the dried sample.…”

“…In all other cases, pre-spiked samples were dried in a speed vac (ThermoSavant SPD1010) and subjected separately to each of three unique acid hydrolyses. Hydrolysis was performed in a similar manner to previously reported studies by Mechref's group [16,17]. In all cases, gas-phase hydrolysis was achieved within a capped glass vial housing the glass autosampler insert.…”

“…Although there are previous literature reports that describe acid hydrolysis of glycoproteins into their constituent monosaccharide units with subsequent analysis using LC-MS [12][13][14][15][16][17] or capillary electrophoresis [18], each of these studies requires derivitization of monosaccharides for detection, likely incorporating measurement bias in proportion to the labeling inefficiency. The current industry standard for analysis of non-derivatized, hydrolyzed sugars -anion exchange chromatography with pulsed amperometric detection [19] (HPAEC-PAD) -is capable of chromatographic resolution of monosaccharides and can be a very sensitive detection technique, but is inherently incompatible with direct mass spectrometry detection, and therefore incompatible with isotope dilution techniques.…”

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

“…in screening newborns for a panel of inborn errors of metabolism) some drugs (11) (e.g. immunosuppressants), and the component analysis of sugars (12).…”

Monitoring Organic Reactions by Micellar Electrokinetic Chromatography

“…It enables the analysis and monitoring of organic reactions in real time [11]. CE is a method which can analyze charged analytes in aqueous solution.…”