Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for the comprehensive characterization of glycoproteins that is able to determine glycan heterogeneity for individual glycosites in addition to providing information about the total N-linked glycan, glycosite-containing peptide and glycoprotein content of complex samples. The NGAG method can also be applied to quantitatively detect glycoprotein alterations in total and site-specific glycan occupancies.

Although, LC-MS is one of the most sensitive and selective analytical techniques, it often suffers from matrix effects, especially when using ESI for analyzing extracts of complicated matrices [1][2][3]. Matrix effects are often caused by the alteration of ionization efficiency of target analytes in the presence of co-eluting compounds in the same matrix. Matrix effects can be observed either as a loss in response (ion suppression) or as an increase in response (ion enhancement). Both the ion suppression and enhancement dramatically affect analytical performance of a method [4]. Therefore, matrix effects must be evaluated when validating an LC-MS method. Since, matrix effects were first observed, efforts have been devoted to understanding the mechanisms and minimizing them [5][6][7][8]. In this commentary article, the causes of matrix effects and methods for evaluating, minimizing and/or compensating for them are discussed. A novel concept, matrix effect factor (MEF) using stable isotopically labeled internal standards (SIL-ISs) is introduced and its application is presented. Since, SIL-ISs are usually added in the very beginning of the sample preparation procedure to compensate for matrix effects and recovery, the MEF reflects any loss/gain from both the sample preparation and 'conventional' matrix effects caused by co-eluting components during ionization in a mass spectrometer (MS) ion source. The causes of matrix effectSince, matrix effects were first observed for ESI MS in 1990s, several mechanisms have been proposed [5,6,9,10]. These include matrix components preventing analyte from gaining access to the charge, competing with analytes to gain charge, interfering with analyte's ability to remain charged in the gas phase, increasing surface tension of droplet or increasing electric resistance. Although, the exact mechanisms of matrix effects are still not fully understood, it has been widely accepted that the co-eluted matrix can alter ionization efficiency of target analytes and influence signal intensity due to the competition for the available charges and for the access to the droplet surface for gas-phase emission during the electrospray process [7,8]. Therefore, any process that changes the ionization efficiency and occurs in the liquid phase and gas phase, will cause matrix effects. For example, some studies showed that the presence of interfering compounds at a higher concentration could increase the viscosity and the surface tension of the droplets, which change the efficiency of their formation and evaporation. The changes in liquid phase could result in the alteration of the amount of charged ions in the gas phase. In addition, matrix components or mobile-phase additives that act as ion-pairing reagents usually reduce ionization efficiency and result in low response [9].Based on the available literature and our experiences, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) sources have less matrix effects than an ESI source, because APCI utilizes gas-ph...

Glycosylation is one of the most common protein modifications and is involved in many functions of glycoproteins. Investigating aberrant protein glycosylation associated with diseases is useful in improving disease diagnostics. Due to the non-template nature of glycan biosynthesis, the glycans attached to glycoproteins are enormously complex; thus, a method for comprehensive analysis of glycans from biological or clinical samples is needed. Here, we describe a novel method for glycomic analysis using glycoprotein Immobilization for glycan extraction (GIG). Proteins or peptides from complex samples were first immobilized on solid support, and other non-conjugated molecules were removed. Glycans were enzymatically or chemically modified on solid-phase before releasing from glycoproteins/glycopeptides for mass spectrometry analysis. The method was applied to the glycomic analysis of both N- and O-glycans.

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two-dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer. Molecular & Cellular

The analysis of sialylated glycans is critical for understanding the role of sialic acid in normal biological processes as well as in disease. However, the labile nature of sialic acid typically renders routine analysis of this monosaccharide by mass spectrometric methods has been difficult. To overcome this difficulty we pursued derivatization methodologies, extending established acetohydrazide approaches to aniline-based methods, and finally to optimized p-toluidine derivatization. This new quantitative glycoform profiling method using MALDI-TOF in positive ion mode was validated by first comparing N-glycans isolated from fetuin and serum and was then exploited to analyze the effects of increased metabolic flux through the sialic acid pathway in SW1990 pancreatic cancer cells by using a co-labeling strategy with light and heavy toluidine. The latter results established that metabolic flux, in a complementary manner to the more well-known impact of sialyltransferase expression, can critically modulate the sialylation of specific glycans while leaving others virtually unchanged.