Biomedical subjects

Shiaw-Lin Wu

Publications and source records attributed to Shiaw-Lin Wu.

14 recordsLinked to original sources

PubMed2006

Glycation of interferon-beta-1b and human serum albumin in a lyophilized glucose formulation. Part III: application of proteomic analysis to the manufacture of biological drugs.

Glycation of interferon-beta-1b and human serum albumin was identified in a lyophilized glucose formulation by a sensitive LC-MS approach. The extent of glycation was measured by a label-free quantitation strategy. The glycation sites were determined by the accurate mass (FTICR MS) with MS/MS measurements on the corresponding tryptic peptides. The extent of glycation was measured by the ratio of the peak intensity between the glycated and the average value for three non-glycated peptides in the same run. Residues lysine 18 of interferon-beta-1b, and lysine 51, lysine 233, and lysine 545 of human serum albumin were more prone to be glycated than other sites in this lyophilized glucose formulation. Residues of lysine 51 and lysine 233 but not lysine 545 of human serum albumin are highly accessible to solvent as found in a solution storage study by Lapolla et al. The extent of glycation of both proteins and the number of glycation sites of human serum albumin were increased with the storage time at 25 degrees C. In total, two glycation sites of interferon beta-1b and 17 glycation sites of human serum albumin were identified in the lyophilized glucose formulation with a storage time at 25 degrees C of 35 days. Among the 17 glycation sites, only lysine 525 of human serum albumin has been found in vivo in diabetic patients by Shaklai et al. As expected, there was no glycation found on both interferon-beta-1b and human serum albumin in the control samples (similar lyophilized formulation but using mannitol instead of glucose).

Amino Acid Sequence↗

PubMed2006

Dynamic profiling of the post-translational modifications and interaction partners of epidermal growth factor receptor signaling after stimulation by epidermal growth factor using Extended Range Proteomic Analysis (ERPA).

In a recent report, we introduced Extended Range Proteomic Analysis (ERPA), an intermediate approach between top-down and bottom-up proteomics, for the comprehensive characterization at the trace level (fmol level) of large and complex proteins. In this study, we extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, epidermal growth factor receptor (EGFR), upon stimulation. Specifically A 431 cells were stimulated with epidermal growth factor after which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2, and 10 min as well as 4 h. High sequence coverage was obtained (96%), and methods were developed for label-free quantitation of phosphorylation and glycosylation. A total of 13 phosphorylation sites were identified, and the estimated stoichiometry was determined over the stimulation time points, including Thr(P) and Ser(P) sites in addition to Tyr(P) sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site were quantitated. No change in the extent of glycosylation with stimulation was observed as expected. Finally potential binding partners to EGFR were identified based on changes in the amount of protein pulled down with EGFR as a function of time of stimulation. Many of the 19 proteins identified are known binding partners of EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets. The detailed molecular information will prove useful in future studies in tissue.

Amino Acid Sequence↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2004

Using LC-MS with de novo software to fully characterize the multiple methylations of lysine residues in a recombinant fragment of an outer membrane protein from a virulent strain of Rickettsia prowazekii.

The outer membrane protein B (OmpB) of the typhus group rickettsiae is an immunodominant antigen and has been shown to provide protection against typhus in animal models. Consequently, OmpB is currently being considered as a potential rickettsiae vaccine candidate to be used in humans. The OmpB from virulent strains are heavily methylated while the attenuated strains are hypomethylated. Western blot analysis of partially digested OmpB revealed that one of the reactive fragments was located at the N-terminus (fragment A, aa 33-272). Recently, we have over expressed, purified, and chemically methylated the recombinant fragment A from Rickettsia prowazekii (Ap). The methylated Ap was thoroughly characterized by LC/MS/MS on the ProteomeX workstation. The protein sequence of Ap with and without methylation was 87.7% and 100% identified, respectively. This high sequence coverage enabled us to determine the sites and extent of methylation on the lysine residues in Ap. All the lysine residues except the C-terminus lysine were either mono-, di- or tri-methylated. In addition, carbamylation on the N-terminus glycine was identified using a combination of denovo sequencing (DeNovoX) and the pattern recognition (SALSA) program with accurate mass measurement. We demonstrated that the use of peptide identification (SEQUEST) in combination with SALSA and denovo sequencing provided a useful means to characterize the sequence and posttranslational modifications of given proteins.

Amino Acid Sequence↗

PubMed2004

A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer.

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.

Amino Acid Sequence↗

PubMed2003

An approach to the proteomic analysis of a breast cancer cell line (SKBR-3).

This report describes the profiling of proteins in a sample prepared by laser capture microdissection (LCM) from a breast cancer cell line (SKBR-3). This experimental approach serves as a model system for proteomic studies on selected tissue samples and for studies of specific cell types. The captured cells were isolated in a dehydrated and reduced state and solubilized with a denaturing buffer. After dilution the protein mixture was digested with trypsin and the resulting peptide mixture was fractionated by reversed phase HPLC (RPLC) and analyzed on an ion trap mass spectrometer. A key part of this study is the combination of the LCM process with an extraction/digestion procedure that allowed effective solubilization of a significant part of the cellular sample in a single step. The identity of the peptides was determined by tandem mass spectrometry measurements in which the resulting spectra were compared with genomic and proteomic databases and protein identifications were made. While only peptides with a high probability assignment were used, the interpretation of mass spectral fragmentation patterns were also confirmed by manual interpretation of the spectra. Also, for the more abundant proteins the initial protein assignment from the best match peptide was strengthened by the observation of additional confirmatory peptide identifications. Another selection criteria was correlation of the mass spectrometric studies with clinical and genomic studies of potential cancer markers in tumor samples. This proteomic study allowed identification of the following proteins: human receptor protein kinase HER-2 or ERBB-2 and related kinases HER-3 and HER-4, the gene products from breast cancer type I and II susceptibility genes and cytoskeletal components such as cytokeratins 8, 18 and 19. Other proteins include fibroblast growth factor receptor variants (FGFR-2&4) and T-lymphoma invasion and metastasis inducing protein 1 (TIAM1). In addition several nonreceptor protein kinases YES, FAK and JAK-1 and 3 were identified. Since the study was performed on a limited number of cells (approximately 10,000) it raises the possibility of such studies being performed on individual patient samples prepared by needle biopsy.

Biomarkers, Tumor↗

PubMed2002

The challenges of developing a sound proteomics strategy.

This paper will review the challenges of developing a proteomics strategy. A key issue is the integration of the two-dimensional (2-D) gel platform with mass spectrometry measurements. The use of both matrix-assisted laser/desorption ionization (on off-line coupling) and electrospray (on-line) ionization are complementary. While the use of one-dimensional and 2-D gels are essential to many aspects of proteomics research (sample preparation, preliminary fractionation and quantitation, storage of protein components), the emergence of shotgun sequencing based on high performance liquid chromatography and tandem mass spectrometry offers a powerful new approach. The latter has particular utility in the characterization of low level samples and complex post-translational modifications. The development of capillary columns, such as 75 to 150 micron, that can be packed in a reproducible manner has been a key step in the development of high sensitivity liquid chromatography/mass spectrometry analysis.

Chromatography↗

PubMed2002

Identification of N-linked oligosaccharides of rat insulin-like growth factor binding protein-4.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. Whereas IGFBP-1 and IGFBP-2 are not glycosylated, IGFBP-3 and IGFBP-4 are N-glycosylated and IGFBP-5 and IGFBP-6 are O-glycosylated. In this study we identified the glycosylation of IGFBP-4 using a nanoflow LC/MS/MS techniques. Although N-linked oligosaccharides are structurally diverse, their variants are well reported in the literature. Based on the molecular weight of the possible oligosaccharide moieties, we identified five different glycosylation isoforms of the protein. Identified glycans were biantennary and differ in the number of sialic acid terminal residues and/or core modification with fucose.

Amino Acid Sequence↗

PubMedDate not supplied

Proteomic profiling of Escherichia coli proteins under high cell density fed-batch cultivation with overexpression of phosphogluconolactonase.

In this study, we used proteomics to better understand the growth on glucose of Escherichia coli in high cell density, fed-batch cultures and the response to overexpression of plasmid-encoded 6-phosphogluconolactonase (PGL). Using liquid chromatography coupled to electrospray mass spectrometry, at least 300 proteins were identified in the cytosolic fraction of the six time points used to monitor the fermentation. The relative abundance changes of selected proteins were obtained by comparing the peak area of the corresponding peptides at a particular m/z (mass over charge ratio) value. During the time course of samples collected during the rapid growth achieved under batch and fed-batch conditions, both the control and recombinant E. coli strains showed up-regulation of proteins participating in the tricarboxylic acid (TCA) cycle, particularly acetyl-CoA synthetase (AcCoAS), malate dehydrogenase (MDH), and succinyl-CoA synthetase (SuccCoAS). In the recombinant strain culture, fumarase was up-regulated until 35 h after inoculation but was not in the control strain culture. In addition, the proteomic measurement detected up-regulation of three well-characterized binding transport proteins in both control and recombinant strains. The up-regulation of TCA cycle enzymes is consistent with the increase in growth rate observed in the cell culture. In addition, up-regulation of these proteins demonstrated the importance of both the pentose-phosphate shunt and TCA cycle to the increased biosynthetic activity required by a high level protein synthesis. This study shows the potential of proteomics using shotgun sequencing (LC/MS of tryptic digests) to measure global changes in protein abundance during a fermentation process and will facilitate the development of robust manufacturing systems.

Bioreactors↗

PubMedDate not supplied

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS).

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.

Animals↗

PubMedDate not supplied

Targeted proteomics of low-level proteins in human plasma by LC/MSn: using human growth hormone as a model system.

This paper describes the profiling of human growth hormone (hGH) in human plasma in order to assess the dynamic range of the ion-trap mass spectrometer for proteomic studies of complex biological samples. Human growth hormone is an example of a low-level plasma protein in vivo, present at subfemtomole levels. This study was performed on a plasma sample in which hGH has been spiked at 10-fold above the natural level, that is approximately 16 pg/microL of plasma. Initially, the measurement was carried out without any sample enrichment and consisted of the following steps: the full set of plasma proteins were reduced, alkylated, and digested with trypsin, and the resulting peptides were separated on a capillary C-18 column and then detected by ion-trap mass spectrometry (1D LC/MS). In addition, this study provided a global view of the serum proteome with over 200 plasma proteins being preliminarily identified. In the MS/MS analysis, hGH was detected by characterization of the first tryptic peptide (T1). The initial identification was confirmed by alternative approaches, which also allowed the evaluation of different sample purification protocols. First, the plasma sample containing hGH was fractionated on a reversed-phase HPLC column and digested, and hGH could now be identified by MS/MS measurements of two tryptic peptides (T1 and T4) by the same 1D LC/MS protocol. In addition, the assignment of peptide identity was made with higher certainty (as measured by an algorithm score). The plasma sample was also fractionated by 1D and 2D gel electrophoresis, the selected bands were digested and analyzed again by the 1D LC/MS protocol. In both cases using the gel prepurifications, hGH was identified with additional peptides. Finally, the plasma sample was analyzed by 2D chromatography (ion exchange and reversed phase) on a new instrumental platform (ProteomeX), and hGH was identified by the observation of five tryptic peptides. In conclusion, these experiments were able to detect growth hormone in the low femtomole level with a dynamic range of 1 in 40 000 by several independent approaches. The amount of growth hormone, while 10-fold above normal in vivo levels, represents concentrations that may be present in disease states (such as acromegaly) and also in doping control measurements. These studies have demonstrated that shotgun sequencing approaches (LC/MS/MS) not only can profile high-abundance proteins in complex biological fluids but also have the potential to identify and quantitate low-level proteins present in such complex mixtures without extensive prepurification protocols. A key to such studies, however, is to use targeted approaches that reduce the complexity of the solute mixture that is presented to the mass spectrometer at a given time point. The various sample preparation protocols described here all improved the quality of the hGH measurement, although in this study the 2D chromatographic approach gave the greatest sequence coverage.

Calibration↗

PubMedDate not supplied

Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS.

This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).

Amino Acid Sequence↗

PubMedDate not supplied

Evaluation of shotgun sequencing for proteomic analysis of human plasma using HPLC coupled with either ion trap or Fourier transform mass spectrometry.

This paper reports on studies directed to the characterization of the proteome of human plasma by the shotgun sequencing approach, namely the use of HPLC coupled to mass spectrometry (MS). The report will present data from two laboratories that allows the comparison of peptide and protein identifications by either accurate mass measurement on a Fourier transform mass spectrometry or MS/MS fragmentation on an ion trap mass spectrometer. Because the dynamic range of the protein components of plasma is one of the largest for a biological sample, the analysis of such a challenging sample was aided by the use of these two MS approaches. The major classes of proteins observed were transport proteins, enzymes, and enzyme inhibitors, blood-clotting factors, membrane-associated proteins including soluble forms of receptors, hormones, immunoglobulins, and other glycoproteins. The protein identifications were also highly consistent with results obtained from 2D gel studies, although a larger number of additional proteins were observed with the shotgun sequencing approach. The quantitation of low to medium level proteins was explored in the ion trap with an add-back of a known amount of human growth hormone (hGH) at a clinically relevant level (5 ug/L). The isotope coded affinity tag (ICAT) approach was used to quantitate successfully different levels of hGH in replicate analysis via the disulfide linked tryptic peptide (T6-T16). These studies suggest that the shotgun sequencing approach can be used to characterize part of the plasma proteome and serve as a starting point for the use of multidimensional analytical approaches for the analysis of complex biological samples.

Algorithms↗

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↗