Biosynthesis of L-Selectin Ligands:  Sulfation of Sialyl Lewis x-Related Oligosaccharides by a Family of GlcNAc-6-sulfotransferases

Phosphorylation and sulfation are two important biological modifications present in carbohydrates, proteins, and glycoproteins. Typically, sulfation and phosphorylation cause different biological responses, so differentiating these two functional groups is important for understanding structure/function relationships in proteins, carbohydrates, and metabolites. Since phosphorylated and sulfated compounds are isobaric, their discrimination is not possible in routinely utilized mass spectrometers. Thus, a novel mass spectrometric method to distinguish them has been developed. Herein, we utilize basic peptides as ion-pairing reagents to complex to phosphorylated and sulfated carbohydrates via noncovalent interactions. By performing ESI-MS/MS on the ion-pair complexes, the isobaric compounds can be distinguished. This is the first study demonstrating that ion-pairing can be used for the detection of phosphorylated compounds and the first study to use ion-pairing in conjunction with MS/MS to obtain structural information about the analytes. ( [2][3][4]. They are also involved in glycolysis processes in the extracellular matrix [7,8]. Sulfation effects protein recognition and helps facilitate clearance of proteins and metabolites from the body [9 -14]. Specifically, sulfated carbohydrates represent a class of biologically active and pharmaceutically important molecules [10]. They are essential in neuropathology and are used as therapeutic agents [9]. Because phosphorylation and sulfation have been linked to different biological functions, differentiating these structures is essential for understanding structure/function relationships in proteins, carbohydrates, and metabolites.Numerous studies [1-4, 6, 7, 9 -18] have been utilized to characterize phosphorylation and sulfation. These methods indicate that selective and sensitive detection of phosphorylated and sulfated compounds in complex matrices is paramount. Radiolabeling [3,6,[11][12][13][14] is one of the commonly used methods to identify phosphorylated and sulfated carbohydrates with good specificity. While this method is hazardous and time-consuming, phosphorylated or sulfated species can be selectively discriminated in the presence of many other compounds in the matrix, using radiolabeling.Mass spectrometry, which is highly selective and sensitive, could possibly provide information about phosphorylation and sulfation without the need for radiolabeling [3,[15][16][17]. Recent MS experiments to identify phosphorylated compounds include exact mass differentiation [2] and discrimination using ionmolecule reactions [1] by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). While these two methods are useful in differentiating the presence of phosphorylated and sulfated compounds, only a few groups have access to these sophisticated instruments. Thus, developing the mass spectrometric methods on more readily available instruments will benefit investigators who are interested in this field.Herein, we utilize basic peptides [9, 19 -23] as i...

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confidence: 99%

A novel mass spectrometric method to distinguish isobaric monosaccharides that are phosphorylated or sulfated using ion-pairing reagents

Zhang

Jiang

et al. 2005

“…In previous studies, sulfated glycans were characterized through radioactive isotope labeling [2,15,16] and nuclear magnetic resonance (NMR) spectrometry [17,18]. These approaches either involve hazardous reagents or suffer from inadequate sensitivity.…”

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confidence: 99%

Sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements

Lei

Novotný

Mechref

2010

Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono-and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components. (J Am Soc Mass Spectrom 2010, 21, 348 -357)

“…Changes in glycosaminoglycan sulfation have been linked to arthritis and other disease states [2][3][4]. Sulfation also is prevalent on cell-surface oligosaccharides in virtually all mammalian tissues, and its presence is fundamental in cellular communication events [5]. Additionally, sulfation is a common modification of glycoproteins, including erythropoeitin [6], mucins [7,8], and the gonadotropin hormones, lutropin, follitropin, and thyrotropin [9].…”

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confidence: 99%

“…When present in glycoproteins, sulfation can alter biological recognition [10,11] and/or facilitate rapid clearance of the protein from the body [12]. Because sulfation is a pervasive, functionally significant biological modification, analytical methods that can monitor its presence are required to better understand its role in protein function, in cell signaling events, and in diseases where sulfation patterns are altered, like arthritis [2,3] and cystic fibrosis [5,7,8].…”

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confidence: 99%

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Enhanced detection of sulfated glycosylation sites in glycoproteins

Jiang

Irungu

Desaire

2005

We demonstrate a method that enhances the mass spectral signal of mono-and disulfated glycopeptides, present in glycoproteins that contain many other nonsulfated glycoforms. This method utilizes the tripeptide Lys-Lys-Lys as an ion-pairing reagent to complex selectively to sulfated species, and enhance their ion signal. The method is applied to the analysis of glycopeptides released from the enzymatic digestion of ovine luteinizing hormone. In this analysis, a disulfated glycopeptide is identified that was previously not detectable by MS assays, and a monosulfated glycoform, present at less than 1% abundance, is identified without any separation or enrichment of these species prior to analysis. In addition to enhancing the ion signal of sulfated glycopeptides, the ion-pairing technique is useful in obtaining structural information about the sulfated species. (J Am Soc Mass Spectrom 2005, 16, 340 -348)