Professor Barry L. Karger's Research Group
(click here for Prof. Karger's biosketch)
A current priority in protein analysis is the comprehensive analysis of larger (>200 KDa) proteins -- quantitatively characterizing heterogeneity in posttranslational modifications, including sites and sequences of glycosylation. Equally important is the capability to do this at trace levels, using separation methods that both provide higher efficiency to resolve similar species, and lower limits of detection in mass spectrometric analysis. Other recent projects of the laboratory have focused on optimizing many different aspect of the proteomic workflow, including sample preparation, high-resolution LC separations, interfaces to mass spectrometry, mass spectrometric methods, data processing, database searching, and validating biomarkers. Applications include the analysis of biopharmaceuticals and biosimilars, and the discovery of biomarkers for cancer in either tissues or blood.
Contents:
Porous Layer Open Tubular (PLOT) Columns
Electron Transfer Dissociation (ETD) in Characterization of Protein Modifications
Extended Range Proteomic Analysis (ERPA)
Monolithic Capillary Columns Improve LC-MS Sensitivity
Collection and Preparation of Trace Samples: LCM, derivatization.
New Data Processing Algorithms and Software
Publications
other recent projects
Porous Layer Open Tubular Columns (PLOT)
 Because ESI-MS is concentration-sensitive rather than mass sensitive, greatly improved limits of detection in LC-MS have been obtained by using narrow-bore columns. The high back-pressure of packed capillary columns motivated the development of monolithic capillary columns (below), and further improvements have been obtained with ultranarrow bore columns having the stationary phase in a porous layer on an open tube.
(overview and references)
Use of Electron Transfer Dissociation (ETD) in Comprehensive
Analysis of Protein Postranslational Modifications
Overview
Full Paper
Extended Range Proteomic Analysis (ERPA)
A significant recent advance is the ERPA (extended range proteomic analysis) platform, which has demonstrated high (>95%) sequence coverage of large complex proteins including sites of phosphorylation and glycosylation, with characterization of the attached glycans. Sensitivity is currently at the 200 fmol level, and improved sample preparation methods are under development. The method involves a combination of digestion with an enzyme that cuts less frequently than trypsin, , e.g. Lys-C, and a novel data acquisition strategy using a hybrid FTMS LTQ-spectrometer, acquiring collision-induced dissociation MS/MS spectra in the linear ion trap concurrently with a high-resolution FTMS scan capable of resolving the high charge states of peptides up to 10 kDa.
Overview by Shiaw-Lin Wu;
Full Paper (J. Proteome Research ASAP Article)
Top-Down Proteomic Analysis of hGH using hybrid LTQ-FT
Paper (Wu et al., Rapid Commun Mass Spectrom., 2004)
Monolithic Capillary Columns Improve LC-MS Sensitivity
In the coupling of liquid chromatography to electrospray mass spectrometry (LC-ESI MS), Dr. Karger's group introduced ultranarrow bore (20 µm i.d.) monolithic columns for high resolution/high sensitivity analysis. By taking advantage of the analytical characteristics of ultra-low flow (10 nL/min) electrospray MS, sensitivity is enhanced for post-translationally modified protein and peptides (e.g. glycosylated and/or phosphorylated) and for hydrophilic components, relative to normal flow rates of 100 – 300 nL/min. Detection limits have recently been improved from the low attomole level with a conventional ion trap to the zeptomole level with the new linear ion traps, a level important for detecting trace proteins in biological matrices.
Monolithic Capillary LC Columns Overview by Jian Zhang
- Ultra-narrow Bore Column Technology for Proteome Research Poster (Zhang, ASMS 2005)
- Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns Paper (Anal. Chem. 2003)
- High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry Paper (Electrophoresis 2003 )
- High-Efficiency, High-Sensitivity Peptide Analysis with Reversed-Phase Nano-LC Monolithic Columns Coupled to ESI-MS Poster Abstract (Ivanov ASMS 2003)
Microchip Integrated Separation Systems for Proteomic Applications
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Multiple open-channel electroosmotic pumping system for microfluidic sample handling. Abstract Paper (Anal Chem, 2003)
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Microfluidic device for capillary electrochromatography-mass spectrometry Paper (Electrophoresis, 2003)
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A miniaturized multichamber solution isoelectric focusing device for separation of protein digests. Paper (Electrophoresis. 2002)
Collection and Preparation of Trace Samples: LCM, derivatization.
Proteomic Study of Breast Cancer Using Laser-Capture Microdissection
Derivatization of Peptides Overview by Anna Pashkova
- Alexa Fluor 350 Increases Number of Peptide and Protein Identifications Paper (Anal. Chem. 2005); Poster Abstract (ASMS 2004)
- Coumarin Tags Enhance MALDI MS Signal Intensities Paper (Anal. Chem. 2005); Poster Abstract (ASMS 2003)
LC/MS Analysis of Tyrosine Phosphorylation using 2-Stage Immunoaffinity Enrichment of pY Proteins and pY Peptides Poster Abstract (Zeck ASMS 2004)
Data and Signal Processing Algorithms and Software for LC-MS and MS/MS
- SeMoP: a new algorithm for unrestricted search for peptide modifications, developed by visiting scientist Christian Baumgartner (pubmed)
- GlyDB: a tool for automatic selection and annotation of glycopeptide MS/MS spectra in LC-MS analysis of protein digests (Jian Min Ren) (pubmed)
- Overview of MEND denoising, PRESEL precursor selection, and denoising by wavelet transforms (Victor Andreev)
- A New Algorithm for Quantitation of LC-MS Proteomic Data Full Poster (Andreev, ASMS 2005)
- MEND: denoising and peak picking algorithm for LC-MS based on matched filtration in the chromatographic time domain Paper; Poster Abstract; Full Poster (Andreev, ASMS 2003)
- PRESEL: Optimized Selection of MS/MS Precursors in LC-MALDI TOF/TOF of Complex Proteomic Mixtures Poster Abstract Full Poster
- Increased Peptide Identifications by Enhanced Data Processing of MALDI MS Data Prior to Database Searching Paper (Rejtar, Anal Chem. 2004) Poster Abstract (Rejtar, ASMS 2004)
- On the Advantage of Denoising and Peak Picking by MEND in LC-MALDI-QqTOF Analysis Abstract Full Poster (Ens, ASMS 2004)
- The Barnett Computer Cluster
The Barnett Linux Cluster
Instrumentation, Karger Group. (Barnett Institute Instrumentation here)
Linear Ion Trap (LTQ) with electron transfer dissociation (ETD) source
Thermo Electron LTQ-FT: Hybrid Linear Ion Trap-Fourier Transform MS
Applied Biosystems AB 4700 TOF/TOF MS
Thermo Electron LTQ Linear Ion Trap
Thermo Electron LCQ DecaXP : 3D ion trap
Thermo Electron LCQ Classic : 3D ion trap
Personnel (Directory)
Research Associate Professor
Shiaw-Lin (Billy) Wu
Research Assistant Professor
Tomas Rejtar
Principal Research Scientists
Roger Kautz
Research Scientists
Shujia (Daniel) Dai
Postdoctoral Fellows
Sangwon Cha
Graduate Students
Zhenke (Jack) Liu
Dipak Thakur
Dong-dong Wang
Zhouxi Wang
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