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Professor
Barry L. Karger's Research Group
Dr.
Karger leads a research program aimed at developing new technologies
to extract as much information as possible from the exceedingly
complex mixtures of naturally occurring proteomic samples.
This research advances all aspects of the proteomic workflow,
including sample preparation, high-resolution LC separations, interfaces
to mass spectrometry, mass spectrometric methods, data processing,
and database searching.
Contents:
Extended
Range Proteomic Analysis (ERPA)
Monolithic Capillary Columns Improve LC-MS
Sensitivity
Collection and Preparation of Trace
Samples: LCM, derivatization.
LC-MALDI-MS:
Multidimensional LC-MS .
New Data Processing Algorithms and
Software
Applications: Proteome Analysis of Methanosarcina acetivorans
Instrumentation
Publications
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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.
Extended
Range Proteomic Analysis (ERPA)
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
-
Multiple
open-channel electroosmotic pumping system for microfluidic
sample handling. Abstract
Paper
(Anal Chem, 2003)
-
Microfluidic
device for capillary electrochromatography-mass spectrometry
Paper
(Electrophoresis, 2003)
-
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)
LC-MALDI-MS:
Multidimensional LC-MS
The
integration of LC with MALDI-TOF MS has been advanced in several
ways. Improved data processing of LC-MS 2D spectra can
remove chemical noise, greatly enhancing signal-to-noise ratios
of low abundance components.
Combined with algorithms for optimizing a precursor ion list
for MS/MS analysis, we can identify over 20% more unique proteins
than using standard data processing approaches.
To increase the throughput of MALDI-TOF MS, we have built
a MALDI spectrometer utilizing a laser with a 2 kHz repetition-rate
laser, over 10-fold faster than commercial (10-300 Hz) instruments.
Its data acquisition rate of 2 samples per second cuts
the MS analysis time from 4.5 hours to 12 minutes, per plate of
700 samples. When used with
a system built to multiplex multiple LC separations into
parallel depositions on a MALDI plate, fast MALDI significantly
increases LC-MS throughput relative to on-line (ESI) methods.
Coupling
of High-Resolution CE and LC to MALDI-MS
Overview by Tomas
Rejtar
- High-speed,
high-resolution monolithic capillary LC-MALDI MS using an off-line
continuous deposition interface for proteomic analysis.
Paper
(Anal Chem 2005)
- A New Strategy
for Comprehensive Proteome Analysis Using Parallel Replicate Separation
with Multiplexed LC-MALDI MS Poster
Abstract (Chen ASMS 2004)
- Universal
Deposition Device for Off-line Coupling of LC to MALDI MS and
MS/MS Poster
Abstract (Rejtar ASMS 2004)
- Closely
Spaced External Standards: a Universal Method of Achieving 5 ppm
Mass Accuracy Over the Entire MALDI Plate in Axial MALDI-TOF MS
Paper
(Moskovets RCMS 2003) Poster
(ASMS '02)
- Comprehensive
Proteome Analysis by Multi-Dimensional Separation Coupled to High
Mass Accuracy MALDI-MS and MALDI-MS/MS Poster
Abstract (Chen, ASMS 2003)
High-Throughput
2 kHz LC-MALDI-TOF Spectrometer Summary
by Eugene Moskovets
- Rapid Quantitative
Analysis of Selected Serum Glycoproteins Using Isotopically Labeled
Peptides By 2 kHz LC -MALDI MS Full
Poster
- MALDI TOF-MS
System with High-Repetition Rate Laser for Fast Analysis of Multiplexed
Separated Peptide Mixtures Poster
Abstract (Moskovets ASMS 2004)
- MALDI-TOF
MS with 2-KHz Laser for Fast Analysis of Separated Peptide Digests
Poster Abstract
(Moskovets ASMS 2003)
Signal Processing Algorithms and Software for LC-MS and MS/MS
- 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
Proteome
Analysis of the archeabacterium Methanocarcina acetivorans C2A
Overview
(Lingyun Li)
High-throughput
Microcoil NMR of Compound Libraries Using Zero-Dispersion Segmented
Flow Analysis Paper
(Kautz, J. Comb. Chem., 2005)
Instrumentation,
Karger Group. (Barnett Institute Instrumentation here)
Thermo Electron LTQ-FT: Hybrid Linear Ion Trap-Fourier Transform
MS
2 kHz MALDI-TOF Spectrometer,
built in-house
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
People
(Directory)
Research
Assistant Professor
Tomas Rejtar
Principal
Research Scientists
Viktor Andreev
Roger Kautz
Eugene Moskovets
Shiaw-Lin (Billy) Wu
Research
Scientists
Enrique Arevalo
Guihua (Eileen) Yue
Postdoctoral
Fellows
Yiqing Lin
Shujia
(Daniel) Dai
Graduate Students
Ye Gu
Lingyun Li
Christine Orazime
Dong-dong Wang
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