Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.     

Biological activity and structural stability of N-deglycosylated recombinant human erythropoietin

Controversy exists regarding the functional role of N-linked oligosaccharides in the hormone erythropoietin. We have now examined the role of carbohydrates in the hormone's action using quantitative enzymatic deglycosylation. N-deglycosylated hormone exhibited full biological activity and potency in vitro. Denaturing with 6M urea and renaturing revealed that both the native and N-deglycosylated forms recovered full activity as long as the intrachain disulfide bonds remained intact. Therefore, receptor recognition, subsequent biological activity and maintenance of tertiary structure are intrinsic properties of the polypeptide chain of erythropoietin.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Production of recombinant human erythropoietin from Pichia pastoris and its structural analysis

AIMS: To design and investigate a recombinant expression system producing a therapeutically important glycoprotein, human erythropoietin (rHuEPO), by Pichia pastoris. METHODS AND RESULTS: EPO cDNA was cloned into pPICZalphaA for expression under control of AOX1 promoter and fused, on the amino-terminal end, with a polyhistidine tag for rapid purification. A target site for factor Xa protease was also introduced, such that cleavage in vitro produced a mature form of rHuEPO having the native N- and C-termini. RHuEPO was characterized as to the extent and nature of N-linked glycosylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and western blotting. The rHuEPO produced was approximately 30 kDa. All three N-linked glycosylation sites were occupied dominantly by Man(17)(GlcNAc)(2). N-glycanase-treated rHuEPO purified but not digested with factor-Xa-protease, showed a spectral peak centered about m/z 20400 Da. CONCLUSIONS: The native polypeptide form of human EPO (c. 18 kDa) was obtained for the first time in P. pastoris expression system, after affinity purification, deglycosylation and factor-Xa-protease digestion. The amount of sodium dodecyl sulfate used prior to deglycosylation was found to be crucial in determining the dominant form of glycan in glycoproteins. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel approaches to protein expression and purification system and structural analysis presented, would be important especially for therapeutic proteins expressed in P. pastoris.     

Mass spectrometric characterization of recombinant human interleukins 1 beta

The cDNA coding for amino acid residues 121-269 of human interleukin 1 beta was cloned and expressed at Sclavo Research Center by C. Baldari et al. (1987, EMBO J. 6, 229-234) using Saccharomyces cerevisiae as the host cell. The purified protein showed a very low specific activity when compared to that of a mature recombinant interleukin expressed from Escherichia coli. Preliminary experiments indicated the occurrence of post-translational events in the yeast-derived protein. In an attempt to correlate the structural modifications and the biological activity of recombinant interleukins, the two proteins were characterized by fast atom bombardment mass spectrometry (FAB/MS) following the FAB-mapping procedure. The amino acid sequence of interleukin expressed in E. coli was identical to that expected whereas the mass spectrometric analysis of the recombinant S. cerevisiae protein confirmed the occurrence of covalent modifications. In particular, the asparagine residue at position 7 (numbering follows the mature active protein sequence) was shown to be glycosylated and the two cysteine residues at position 8 and 71 were involved in an S-S bridge. The results demonstrate the value of FAB/MS in the quality control of recombinant interleukins.     

Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin

Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.     

Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin

Physicochemical and biological properties of recombinant human erythropoietin (rhEPO) were compared with human urinary erythropoietin (uEPO). uEPO and rhEPO were purified to apparent homogeneity from the urine of patients with aplastic anemia and from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human EPO, respectively. The microheterogeneous nature of both factors, observed on isoelectric focusing, is derived from the difference of the number of terminal sialic acid residues bound to the carbohydrate chains of the EPO molecule. The primary structure of rhEPO, consisting of 165 amino acid residues, was determined, and the C-terminal arginine predicted from the cDNA sequence was confirmed to be missing, as described previously (Recny et al. (1987) J. Biol. Chem. 262, 17156). Three N-glycosylation and one O-glycosylation sites of both factors were determined as Asn24, Asn38, and Asn83 and Ser126, respectively. Two disulfide linkages are located between Cys7 and Cys161, and between Cys29 and Cys33, in both EPOs. Hematogenic potencies of rhEPO and uEPO compared in normal and in partially nephrectomized rats were approximately the same. Both factors also stimulated the colony formation of CFU-E, BFU-E, and CFU-Meg in a dose-dependent manner. From these results, it is concluded that rhEPO produced in CHO cells transfected with cDNA clone for human EPO is indistinguishable from uEPO physicochemically and biologically, and is valuable for further research and for clinical use.     

Expression and structural characterization of the recombinant human doppel protein

The doppel protein (Dpl) is a newly recognized prion protein (PrP)-like molecule encoded by a novel gene locus, prnd, located on the same chromosome as the PrP gene. To study the structural features of Dpl, we have expressed recombinant human Dpl corresponding to the putative mature protein domain (residues 24-152) in Escherichia coli. The primary structure of the recombinant Dpl 24-152 was characterized using gel electrophoresis, N-terminal Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization mass spectrometry. Dpl 24-152 was shown to contain two disulfide bonds (Cys94-Cys145 and Cys108-Cys140). The secondary structure of Dpl was analyzed using far-UV circular dichroism spectroscopy. Dpl 24-152 was found to be an alpha-helical protein having a high helical content (40%). Dpl 24-152 exhibited characteristics of a thermodynamically stable protein that undergoes reversible and cooperative thermal denaturation. In addition, Dpl was found to be soluble and sensitive to proteinase K digestion. Therefore, Dpl 24-152 possesses biochemical properties similar to those of recombinant PrP. This study provides knowledge about the molecular features of human Dpl that will be useful in further investigation into its normal function and the role it may play in neurodegenerative diseases.     

Mass spectral characterization of organophosphate-labeled lysine in peptides

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9 amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8 amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.     

Mass spectrometry-based proteomic studies of human anaplastic large cell lymphoma

Malignant lymphomas are a diverse group of malignant neoplasms that arise as a result of a complex interplay of multiple factors including genetic aberrations, immunosuppression, and exposure to noxious agents such as ionizing radiation and chemical agents. Anaplastic large cell lymphoma (ALCL) is an aggressive T-lineage lymphoma harboring chromosomal translocations involving the anaplastic lymphoma kinase (ALK) tyrosine kinase. The most common translocation in ALCL is the t(2;5)(p23;q35). This results in the formation of a chimeric fusion kinase, nucleophosmin/ALK. Nucleophosmin/ALK activates numerous downstream signaling pathways resulting in enhanced survival and proliferation. Using a variety of mass spectrometry-driven proteomic strategies, we have studied several aspects of the ALCL proteome. In this review, we provide a summary of mass spectrometry-based proteomic studies that expands the current understanding of the molecular pathogenesis of ALCL and provides the basis for the identification of biomarkers and targets for novel therapeutic agents.     

Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.     

Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.     

Effectiveness of weekly subcutaneous recombinant human erythropoietin administration for chemotherapy-induced anemia

The effects of weekly subcutaneousrecombinant human erythropoietin (r-hEPO)administration on anemia during chemotherapy includingcisplatin and 5-fluorouracil in patients with head andneck carcinomas were examined. Weekly subcutaneousr-hEPO administration in cancer patients has not beeninvestigated previously. Patients were treated withr-hEPO 100 IU/kg (2 patients), 200 IU/kg (6 patients),or 400 IU/kg (5 patients), or placebo, andeffectiveness was evaluated by monitoring hemoglobinconcentration changes after administration for 8weeks. Hemoglobin concentrations in all 3 r-hEPOdosage groups were higher than that in the controlgroup during chemotherapy. All r-hEPO doses producedimprovements in the anemia induced by chemotherapy;however, the 400 IU/kg dose was most effective. Therequirement for blood transfusions decreased inpatients receiving r-hEPO therapy, and no significantside-effects were associated with r-hEPOadministration. These results suggest thatchemotherapy-induced anemia can be prevented by weeklysubcutaneous r-hEPO administration.     

Mass spectrometry-based proteomics for the detection of plant pathogens

Plant diseases caused by fungi, oomycetes, viruses, and bacteria are devastating both to the economy and to the food supply of a nation. Therefore, the development of new, rapid methods to identify these pathogens is a highly important area of research that is of international concern. MS-based proteomics has become a powerful and increasingly popular approach to not only identify these pathogens, but also to better understand their biology. However, there is a distinction between identifying a pathogen protein and identifying a pathogen based upon the detection of one of its proteins and this must be considered before the general application of MS for plant pathogen detection is made. There has been a recent push in the proteomics community to make data from large-scale proteomics experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MS as a universal plant pathogen detection technology.     

Expression and characterization of human N-acetylglucosaminyltransferases and alpha2,3-sialyltransferase in insect cells for in vitro glycosylation of recombinant erythropoietin

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennary-type complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of N-acetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using UDP-14C-Gal and CMP-14C-Sia as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the red blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.