Biomedical subjects

Jeongkwon Kim

Publications and source records attributed to Jeongkwon Kim.

7 recordsLinked to original sources

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↗

PubMed2005

Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics.

Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 microm i.d. x 40-200 cm fused-silica capillaries packed with 1.4-3-microm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of >12 000 different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (approximately 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10(6) based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomics sample.

Automation↗

PubMed2005

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.

Animals↗

PubMed2004

Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry.

In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine beta-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C(18)-bonded stationary phase silica surface.

Animals↗

PubMed2003

High sequence coverage of proteins isolated from liquid separations of breast cancer cells using capillary electrophoresis-time-of-flight MS and MALDI-TOF MS mapping.

A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines. Intact proteins are isolated using multidimensional liquid-phase separations that permit the collection of individual protein fractions. Protein digests are then analyzed by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and by capillary electrophoresis-electrospray ionization (CE-ESI)-TOF MS peptide mapping. These methods can be readily interfaced to the relatively clean proteins resulting from liquid-phase fractionation of cell lysates with little sample preparation. Using combined sequence information provided by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Furthermore, an accurate intact protein MW value (within 150 ppm) can be obtained from ESI-TOF MS. The intact MW together with high coverage sequence information provides accurate identification. More notably the high sequence coverage of CE-ESI-TOF MS together with the MS/MS information provided by the ion trap/reTOF MS elucidates posttranslational modifications, sequence changes, truncations, and isoforms that may otherwise go undetected when standard MALDI-MS peptide fingerprinting is used. This capability is critical in the analysis of human cancer cells where large numbers of expressed proteins are modified, and these modifications may play an important role in the cancer process.

Amino Acid Sequence↗

PubMed2003

Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis.

Several semiempirical models for peptide electrophoretic mobility have been tested for capillary electrophoresis (CE) separation with a positively charged capillary using on-line CE combined with electrospray ionization-mass spectrometry (ESI-MS). In the current system with pH 2.7, the expression q/M(0.56) provided the best correlation with the electrophoretic mobility in the analysis of a set of 18 standard samples, where q is the calculated net charge and M is the molecular weight. The peptides resulting from various digests of horse heart myoglobin or bovine hemoglobin were used to demonstrate the validity of this correlation. Post-translationally modified peptides from tryptic digest of human myelin basic protein were also investigated and were found to provide excellent correlation with the linear plot when the total charge of the peptide was correctly calculated. If the total charge was not properly calculated then the post-translationally modified peptides fell off the linear plot. Using this method five arginine residues (residues 5, 49, 54, 97 and 130) were found to be partially citrullinated, four glutamine residues (residues 8, 103, 121 and 147) were found to be partially deamidated and both methionines at residues 21 and 167 were found to be partially oxidized. Three peptides were found with phosphorylation; TPPPSQGK (residue 98 to 105), FSWGAEGQR (residue 114 to 122), and SGSPMAR (residue 163 to 169) and Arginine at 107 was found to be partially monomethylated or dimethylated. The method may provide an excellent means of identifying the presence of peptides with post-translational modifications in conjunction with MS.

Amino Acid Sequence↗

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↗