Comparison of N-linked Glycoproteins in Human Whole Saliva, Parotid, Submandibular, and Sublingual Glandular Secretions Identified using Hydrazide Chemistry and Mass Spectrometry

INTRODUCTION: Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids. MATERIALS AND METHODS: We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography-tandem mass spectrometry was used to identify the salivary N-glycoproteins. RESULTS: A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions. CONCLUSIONS: Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.     

Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining nonspecific protease digestion, nanoflow liquid chromatography, and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-Pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein, and bovine fetuin using nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC-Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan microheterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.     

N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

Characterization of the human blood plasma proteome

We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

Quantitative N-linked glycoproteomics of myocardial ischemia and reperfusion injury reveals early remodeling in the extracellular environment

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.     

Identification and quantification of N-linked glycoproteins using hydrazide chemistry,stable isotope labeling and mass spectrometry

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.     

Development of a semi-automated LC/MS/MS method for the simultaneous quantitation of 14,15-epoxyeicosatrienoic acid, 14,15-dihydroxyeicosatrienoic acid, leukotoxin and leukotoxin diol in human plasma as biomarkers of soluble epoxide hydrolase activity in vivo

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography-mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid-liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05-50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.     

Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry

Protein glycosylation (e.g., N-linked glycosylation) is known to play an essential role in both cellular functions and secretory pathways; however, our knowledge of in vivo N-glycosylated sites is very limited for the majority of fungal organisms including Aspergillus niger. Herein, we present the first extensive mapping of N-glycosylated sites in A. niger by applying an optimized solid phase glycopeptide enrichment protocol using hydrazide-modified magnetic beads. The enrichment protocol was initially optimized using both mouse blood plasma and A. niger secretome samples, and it was demonstrated that the protein-level enrichment protocol offered superior performance over the peptide-level protocol. The optimized protocol was then applied to profile N-glycosylated sites from both the secretome and whole cell lysates of A. niger. A total of 847 N-glycosylated sites from 330 N-glycoproteins (156 proteins from the secretome and 279 proteins from whole cells) were confidently identified by LC-MS/MS. The identified N-glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, and storage vacuoles, supporting the important role of N-glycosylation in the secretory pathways. In addition, these glycoproteins are involved in many biological processes including gene regulation, signal transduction, protein folding and assembly, protein modification, and carbohydrate metabolism. The extensive coverage of N-glycosylated sites and the observation of partial glycan occupancy on specific sites in a number of enzymes provide important initial information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.     

Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts toward complex-type N-glycosylation in classical galactosemia

A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6.     

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]     

N-glycoproteome Analysis of the Secretome of Human Metastatic Hepatocellular Carcinoma Cell Lines Combining Hydrazide Chemistry,HILIC Enrichment and Mass Spectrometry

Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.     

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30–13.68%) and reproducible (%CV range = 1.49–10.81%) with a LOQ of 2.5 μg/mL.     

The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies

Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC-ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip-MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography-electrospray mass spectrometry and Edman degradation.

An N-linked glycosylation in a novel C-lectin protein from snake venom was observed by Edman degradation and liquid chromatography-electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N-glycosylation site. The result was further confirmed by N-glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high-mannose type by tandem MS coupled with alpha-mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry.     

Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins

We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC–MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.     

Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry

Glycoproteins make up a major and important part of the salivary proteome and play a vital role in maintaining the health of the oral cavity. Because changes in the physiological state of a person are reflected as changes in the glycoproteome composition, mapping the salivary glycoproteome will provide insights into various processes in the body. Salivary glycoproteins were identified by the hydrazide coupling and release method. In this approach, glycoproteins were coupled onto a hydrazide resin, the proteins were then digested and formerly N-glycosylated peptides were selectively released with the enzyme PNGase F and analyzed by LC-MS/MS. Employing this method, coupled with in-solution isoelectric focusing separation as an additional means for pre-fractionation, we identified 84 formerly N-glycosylated peptides from 45 unique N-glycoproteins. Of these, 16 glycoproteins have not been reported previously in saliva. In addition, we identified 44 new sites of N-linked glycosylation on the proteins.     

Quantification of apolipoprotein A-I mimetic peptide D-4F in rabbit plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

The apolipoprotein A-I mimetic peptide D-4F is a potential therapeutical agent effective in maintaining cardiovascular health. A bioanalytical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) to quantitate the D-4F amount in rabbit plasma was developed and validated. A compound with a close structure similarity to the D-4F (only one amino acid A–V altered) was used as an internal standard. Both D-4F and the internal standard were extracted by protein precipitation using acetonitrile/0.2% Triton XL 80N. The correlation coefficient of the calibration curve was 0.9991 in the range 20–40,000 ng/mL. This assay can be used for pharmacokinetic studies of the drug. Also, it may be adjusted for the quantification of other members of apolipoprotein A-I mimetic peptide family.     

Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma.

Plasma from many vertebrates, including pigs, contains a soluble component that inhibits the CO2 hydrase activity of carbonic anhydrase (CA). This activity was purified to homogeneity (approximately 4000-fold) from porcine plasma using a combination of DEAE-Affi-Gel Blue chromatography and carbonic anhydrase II-affinity chromatography, yielding 16 mg of inhibitory protein/L of plasma. This protein, porcine inhibitor of carbonic anhydrase (pICA), is a monomeric protein with an apparent molecular mass of 79 kDa, as determined by electrospray mass spectrometry. As isolated, pICA contains about 3 kDa of N-linked glycosylation removable by peptide N-glycosidase F. pICA inhibits CA reversibly with a 1:1 stoichiometry. pICA is a potent and specific inhibitor of the CA II isozyme, with Ki < 0.1 nM for porcine CA II at pH 7.4. Although the Ki is dependent on the CA isozyme type (CA II < CA IV < CA III approximately CA I), it is relatively insensitive to the species source, as long as it is mammalian. The Ki is pH dependent with log Ki decreasing linearly as the pH decreases, implicating at least one ionizable group with the pKa < or = 6.5 in the binding interaction. The isozyme and species dependence of the inhibition suggest that pICA interacts with amino acids on the surface of CA II.