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

Abstract

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...

Biologically-Inspired Peptide Reagents for Enhancing IMS-MS Analysis of Carbohydrates

“…Although they have yet to be used in IMS studies, Desaire and coworkers have shown that ion-pairing reagents can be used to bind and ionize certain functionalized (e.g., phosphorylated or sulfated) saccharide species [21,22]. Due to the acidity of these functional groups, such ion-pairing reagents are typically oligomers of basic amino acid residues, such as trilysine [22].…”

“…Due to the acidity of these functional groups, such ion-pairing reagents are typically oligomers of basic amino acid residues, such as trilysine [22]. These reagents make it possible to differentiate between phosphorylated and sulfated species based on characteristic fragment ions produced upon collision-induced dissociation [21]. Peptide-based reagents are also appealing because, unlike metal cations, they have inherent structure that may be flexible.…”

Distinguishing phosphorylation and sulfation in carbohydrates and glycoproteins using ion-pairing and mass spectrometry

“…Other MS methods, such as matrix-assisted laser desorption/ionization (MALDI) [21] and electrospray ionization (ESI) [22], have been recently reported to distinguish phosphorylation and sulfation. In MALDI analysis, sulfated and phosphorylated products ionize somewhat differently, and these differences could potentially be used to distinguish phosphate and sulfate in an unknown compound [21].…”

“…In MALDI analysis, sulfated and phosphorylated products ionize somewhat differently, and these differences could potentially be used to distinguish phosphate and sulfate in an unknown compound [21]. In ESI analysis, ion-pairing reagents have been utilized to complex with singly phosphorylated or sulfated monosaccharides [22]. The isobaric ion-pairing complexes can be differentiated in MS/MS experiments because of their different chemical reactivities.…”

“…The isobaric ion-pairing complexes can be differentiated in MS/MS experiments because of their different chemical reactivities. The fundamental study indicates that this approach is only functional group dependent [22] and, thus, it could potentially be effective in discriminating the phosphorylation and sulfation in larger biologically relevant molecules. If the ion-pairing method, which distinguishes phosphorylated and sulfated monosaccharides, could be adapted to larger macromolecules, such as oligosaccharides and glycoproteins, this approach would afford a unique and effective way to distinguish phosphorylation from sulfation.…”