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PubMed2005

Characterisation of protein microheterogeneity and protein complexes using on-chip immunoaffinity purification-mass spectrometry.

Post-translational modification of proteins may influence their interactions with other plasma proteins, as well as having an effect on many aspects of the metabolism of the protein, such as receptor binding, tissue uptake, degradation and excretion. Many post-translational modifications occur in a physiological context, while others are specific for certain diseases, which is why they are of diagnostic importance in clinical proteomics. Analytical approaches to the study of post-translational modifications and protein complexes through the combined use of on-chip immunological affinity purification on a surface-enhanced laser desorption/ionisation platform and subsequent mass spectrometry are illustrated in the author's own work relating to plasma transthyretin (TTR) and retinol-binding protein (RBP). In those studies, both the aspects of post-translational modifications of TTR and the formation of a protein complex between TTR and RBP have been discussed. Such aspects are of diagnostic interest in clinical proteomics, especially with regard to the modification of TTR in relation to the occurrence of amyloidotic diseases.

Amyloid↗
PubMed2006

Sub-speciating Campylobacter jejuni by proteomic analysis of its protein biomarkers and their post-translational modifications.

We have identified several protein biomarkers of three Campylobacter jejuni strains (RM1221, RM1859, and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectra (TOF MS) of bacterial cell lysate supernatants ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were: DNA-binding protein HU, translation initiation factor IF-1, cytochrome c553, a transthyretin-like periplasmic protein, chaperonin GroES, thioredoxin Trx, and ribosomal proteins: L7/L12 (50S), L24 (50S), S16 (30S), L29 (50S), and S15 (30S), and conserved proteins similar to strain NCTC 11168 proteins Cj1164 and Cj1225. The protein biomarkers identified appear to represent high copy, intact proteins. The significant findings are as follows: (1) Biomarker mass shifts between these strains were due to amino acid substitutions of the primary polypeptide sequence and not due to changes in post-translational modifications (PTMs). (2) If present, a PTM of a protein biomarker appeared consistently for all three strains, which supported that the biomarker mass shifts observed between strains were not due to PTM variability. (3) The PTMs observed included N-terminal methionine (N-Met) cleavage as well as a number of other PTMs. (4) It was discovered that protein biomarkers of C. jejuni (as well as other thermophilic Campylobacters) appear to violate the N-Met cleavage rule of bacterial proteins, which predicts N-Met cleavage if the penultimate residue is threonine. Two protein biomarkers (HU and 30S ribosomal protein S16) that have a penultimate threonine residue do not show N-Met cleavage. In all other cases, the rule correctly predicted N-Met cleavage among the biomarkers analyzed. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms for protein/pathogen identification. (5) There were fewer biomarker mass shifts between strains RM1221 and RM1859 compared to strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (and thus underlying genetic variations), this suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782 (in addition, a protein biomarker prominent in the spectra of RM1221 and RM1859 was absent from the RM3782 spectrum due to a nonsense mutation in the gene of the biomarker). These observations were confirmed by a nitrate reduction test, which showed that RM1221 and RM1859 were C. jejuni subsp. jejuni whereas RM3782 was C. jejuni subsp. doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms may easily subspeciate bacterial microorganisms. (6) Finally, the number and variation of PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more possible PTMs than suggested previously in the literature.

Amino Acid Sequence↗
PubMed2005

Overproduction of Pichia pastoris or Plasmodium falciparum protein disulfide isomerase affects expression, folding and O-linked glycosylation of a malaria vaccine candidate expressed in P. pastoris.

Production of recombinant malaria proteins in the methylotrophic yeast Pichia pastoris has been difficult due to constraints in transcription, translation and/or post-translation controls. Use of codon-optimized genes has resolved many of the transcriptional controls; however, efforts to overcome translational and post-translational modifications involving disulfide bond formation and glycosylation have been mostly restricted to knocking-out putative N-linked glycosylation sites. We report now on the effect of overproduction of P. pastoris protein disulfide isomerase (PpPDI) and Plasmodium falciparum (PfPDI) on production of a disulfide-rich P. falciparum transmission-blocking vaccine candidate, Pfs25. Pfs25 is expressed in P. pastoris as two isoforms (A and B); the A form has been selected for Phase I human studies. Overproduction of PpPDI in the P. pastoris Pfs25 production clone markedly enhanced the expression level of Pfs25(A) and (B) by 3-fold, while overproduction of PfPDI increased the proportion of Pfs25(A) compared to (B). The resultant Pfs25 products were purified and fully characterized biochemically. In addition to differences in production levels, the mass spectra of PpPDI-Pfs25(A) compared to Pfs25(A) and PfPDI-Pfs25(A) were different due to the pattern and level of O-linked glycosylation. The overproduction of PpPDI or PfPDI provides new platforms for expression of disulfide-rich malaria proteins.

Animals↗
PubMed2005

Sequence tag scanning: a new explorative strategy for recognition of unexpected protein alterations by nanoelectrospray ionization-tandem mass spectrometry.

Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a2, b2, b3, y1, y2, and y3. The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms.

Acetylation↗
PubMedDate not supplied

Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR).

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.

Amino Acid Sequence↗
PubMed1989

Characterization of isoprenoid involved in the post-translational modification of mammalian cell proteins.

Mammalian cell proteins, modified post-translationally by derivatives of [3H]mevalonic acid, were subjected to methylation and sulfonium salt cleavage reactions previously used to release isoprenoids from cysteine residues in yeast peptides. The labeled isoprenoid extracted into chloroform comigrated with farnesol through a series of chromatography steps including Sep-Pak C-18 fractionation, size exclusion on Bio-Beads, and reverse-phase chromatography. Further resolution of the material by normal phase liquid chromatography and thin layer chromatography demonstrated the presence of farnesol, nerolidol, and other unidentified hydrophobic derivatives. Similar products were generated when S-farnesyl cysteine was subjected to the methylation and cleavage procedures. These preliminary findings suggest that farnesylation of cysteine residues accounts for the well documented incorporation of mevalonic acid into mammalian cell proteins.

Animals↗
PubMed2006

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry.

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.

Acetylation↗
PubMed1995

Protein modification by ADP-ribose via acid-labile linkages.

As substrate for protein-mono-ADP-ribosyltransferases, NAD has been shown to be the donor of ADP-ribose to many different nucleophiles found in proteins. This post-translational modification of proteins has been implicated in the regulation of membrane-associated processes including signal transduction, muscle cell differentiation, and protein trafficking and secretion. Described here is the preparation and chemical characterization of low molecular weight conjugates that were used as models for an acetal linkage between ADP-ribose and the hydroxyl group of a protein acceptor such as serine, threonine, tyrosine, hydroxyproline, or hydroxylysine residues. Model conjugates of ADP-ribose containing an acetal linkage were prepared, their structures were established by NMR, and the chemical stability of the linkage to ADP-ribose was studied and compared to the other known ADP-ribosyl-amino acid linkages. The rapid release of intact ADP-ribose from the acetal model conjugates in 44% formic acid distinguished them chemically from all the other known ADP-ribosyl-amino acid modifications. Rat liver proteins were shown to be modified by ADP-ribose in vivo by acid-labile linkages, providing evidence for a new class of endogenous ADP-ribose modification of animal cell proteins. The amount of modification was approximately 16 pmol of ADP-ribose per mg of total protein, and proteins modified by acid-labile linkages were detected in all subcellular fractions examined, suggesting that the scope of this modification in vivo is broad.

Adenosine Diphosphate Ribose↗
PubMed1997

Post-translational non-enzymatic modification of proteins. II. Separation of selected protein species after glycation and other carbonyl-mediated modifications.

There are two strategies applicable to revealing non-enzymatic post-translational modifications of proteins; while assaying of the hydrolytically stable adducts was the subject of our previous communication [1], here we attempted to review separation technologies for the unfragmented modified proteins. There are a few standard procedures used for this purpose, namely Laemmli gel electrophoresis, different modes of gel permeation chromatography and boronate affinity chromatography. The latter approach makes use of the vicinal hydroxy groups present in glycated proteins. Some (but not all) arising adducts exhibit typical fluorescence which can be exploited for detection. In most cases fluorescence is measured at 370/440 nm for the so-called advanced glycation products or at 335/385 nm for the only so far well characterized glycation marker (pentosidine). Some indication exists that, e.g., synchronous fluorescence detection will probably in the future add to the selectivity and allow the distinction of the different adducts arising during non-enzymatic post-translational modifications (glycation). The proteins reviewed are serum albumin, collagen and lens proteins while glycation of hemoglobin is the subject of another review within the present volume.

Chromatography, Liquid↗
PubMed2005

Fluorescence-based cloning of a protein tyrosine kinase with a yeast tribrid system.

Post-translational modifications of proteins control myriad biological functions. However, relatively few methods exist for the identification of the enzymes that catalyze these modifications. To expand this repertoire, we report a yeast genetic approach that enables the identification of protein tyrosine kinases (PTKs) from cDNA libraries. Yeasts were transformed with four vectors encoding: 1) a potentially universal PTK substrate fused to the LexA DNA binding domain, 2) the Grb2-SH2 domain fused to the B42 activation domain, 3) a fluorescent reporter gene controlled by LexA DNA sites, and 4) a Jurkat cDNA library. Transient expression of PTKs, such as the lymphocyte-specific kinase Fyn, resulted in phosphorylation of the DNA-bound substrate, recruitment of the Grb2-SH2 domain, and activation of the fluorescent reporter gene. This brief induction of protein expression circumvented the potential toxicity of PTKs to the yeast. Fluorescence activated cell sorting (FACS) enabled isolation of PTKs, and these enzymes were further characterized by flow cytometry and immunoblotting. This approach provides a potentially general method for the identification and evaluation of enzymes involved in the post-translational modification of proteins.

Cloning, Molecular↗
PubMed2006

Opportunities for structure-based design of protease-directed drugs.

As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.

Aspartic Acid Endopeptidases↗
PubMed2005

Optimization of DsRed production in Escherichia coli: effect of ribosome binding site sequestration on translation efficiency.

DsRed-Express, a popular reporter protein, cannot be expressed in Escherichia coli using a consensus ribosome binding site (RBS) potentially due to basepairing in the RBS that inhibits translation initiation. Saturation mutagenesis was used to probe for a gene sequence that minimized basepairing in the RBS while maintaining the same spectral properties and maturation characteristics as DsRed-Express. The new DsRed, designated here as RFP(EC) for E. coli optimized red fluorescent protein, fluoresces 2.5 times greater than DsRed-Express when expressed from the same vector.

Binding Sites↗
PubMed2004

Rapid translation system: a novel cell-free way from gene to protein.

Proteome research has recently been stimulated by important technological advances in the field of recombinant protein expression. One major breakthrough was the development of a new generation of cell-free transcription/translation systems. The open and flexible character of these systems allows direct control over expression conditions via the addition of supplements to the expression reaction. The possibility of working with linear expression templates instead of cloned plasmids and the ease of downstream processing, circumventing the need for cell-lysis, makes them ideally suited for high-throughput screening applications. Among these novel cell-free systems, the Rapid Translation System (RTS) developed by Roche is the first one that is scalable from micrograms to milligrams of protein. This review describes the basic principles of RTS which differentiate it from traditional in vitro expression technologies, starting from template generation to high-end applications like labeling for structural biology research. Recent results obtained by RTS users from different institutions are presented to illustrate each step of a novel cell-free protein expression workflow and its benefits compared to traditional cell-based expression.

Cell Extracts↗
PubMed2006

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.

Cell-Free System↗
PubMed2004

Re-examination of the post-translational arginylated protein of 125-kD initially identified as N-STOP.

Post-translational modification of proteins is a complex mechanism by which cells regulate protein activities. One post-translational modification is the incorporation of arginine into the NH2-terminus of proteins. It has been hypothesized that in rat brain extracts, one of the proteins modified by this reaction is the microtubule-associated protein Neuronal Stable Tubule Only Polypeptide (N-STOP). This was inferred from its electrophoretic mobility (125 kD) and because it was immunoprecipitated with a monoclonal antibody against the N-STOP. However, this hypothesis is not supported by our recent results. Herein, we found that rat N-STOP interacts with Ca(2+)-calmodulin, whereas the 125-kD [14C]-arginylated protein does not. The 125-kD [14C]-arginylated protein from rat brain is separated from the N-STOP by two-dimensional electrophoresis, and it is not recognized by a STOP monoclonal antibody. Mouse brain contains N-STOP, which migrates as a protein of 116 kD and could not be labeled by the post-translational incorporation of [14C]-arginine. The 125-kD [14C]-arginylated protein appears in wild-type as well as in STOP knock out mice. Based on these results, we conclude that the 125-kD arginylated protein is different from N-STOP.

Animals↗
PubMed2005

Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells.

Schneider 2 (S2) cells from Drosophila melanogaster have been used as a plasmid-based, non-lytic expression system for foreign proteins. Here, a plasmid encoding the human erythropoietin (hEPO) gene fused with a hexahistidine (His(6)) tag under the control of the Drosophila metallothionein (MT) promoter was stably transfected into Drosophila S2 cells. After copper sulfate induction, transfected S2 cells were found to secrete hEPO with a maximum expression level of 18 mg/L and a secretion efficiency near 98%. The secreted hEPO from Drosophila S2 had an apparent molecular weight of about 23-27 kDa which was significantly lower than a recombinant hEPO expressed in Chinese hamster ovary (CHO) cells (about 36 kDa). N-glycosidase F digestion almost completely eliminated the difference and resulted in the same molecular weight ( approximately 20 kDa) of de-N-glycosylated hEPO proteins. These data suggest that recombinant hEPO from S2 cells was modified with smaller N-glycans. Subsequently, the major N-glycans were identified following glycoamidase A digestion, labeling with 2-aminopyridine (PA), and two-dimensional high-performance liquid chromatography (HPLC) analysis in concert with exoglycosidase digestion. This analysis of N-glycans revealed that hEPO was modified to include paucimannosidic glycans containing two or three mannose residues with or without core fucose. A similar glycosylation pattern was observed on a recombinant human transferrin expressed in S2 cells. These results provide a detailed analysis of multiple N-glycan structures produced in a Drosophila cell line that will be useful in the subsequent application of these cells for the generation of heterologous glycoproteins.

Animals↗
PubMed2007

Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system.

The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that beta-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn(2+)), a cofactor for beta-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn(2+) addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn(2+) addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn(2+) only had this effect late in the culture process. These data indicate that the addition of Mn(2+) has complex effects on stressed batch fed cultures.

Amino Acids↗
PubMed2005

Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells.

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20mg/L) in Sf21 cells.

Animals↗