This proposal will involve the development of new methodology and instrumentation for sequencing of protein digests and post-translatinally modified proteins at the low picomole level and below. This work will be developed specifically for sequencing portions of tumor related target proteins, including 0p 18 and Hsp 27. These proteins will be obtained from tumor cells and isolated using 2-D Gel electrophoresis in the 2-D Gel Laboratory at The University of Michigan. These proteins are obtained at very low levels where they are enzymatically cleaved into peptide fractions for sequencing, but they often can not be sequenced by the Edman method due to enter N-terminal blockage, low amounts of sample or the inability to detect post-translational modifications. This proposal will involve development of on-line capillary separation methods interfacedvia miroelectrospry for detection using an ion trap/reflctron time-of-flight mass spectrometer to achieve the ultimate in sensitivity for detection of the vary low levels of peptides available. These methods include the use of capillary electrophoresis, packed and open tubular capillary electrochromatography and isoelectric focusing methods of separation and high sensitivity detection of peptides. These methods will be interfaced to the IT/reTOP MS for detection where the trapping capabilities of the ion trap and various other instrumental modifications will be used to achieve the required detection levels. In addition, the speed of this hybrid device will be shown to be uniquely suited to achieve full resolution in detection on-line for even the fastest separation and even able to perform on-line MS/MS for structural analysis without loss of resolution. Following separation by capillary methods on-line detection and MS/MS in the trap via resonance methods for isolation and excitation will be used to perform structural analysis of these target proteins. In addition, secondary methodology using capillary separation of protein digests followed by off-line MALDI in the trap produce extensive fragmentation via long term decay which can be used for structural analysis at low levels. These ions decay over periods of milliseconds in the trap but are detected as stable fragment ions in the TOF detector. Ultimately these complementary methods will be used to generate structural information on the sequence and modifications of these cellular proteins at the picomole or subpicomole level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM049500-06S1
Application #
6344275
Study Section
Special Emphasis Panel (ZRG3 (18))
Program Officer
Edmonds, Charles G
Project Start
1994-09-12
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
6
Fiscal Year
2000
Total Cost
$38,986
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Surgery
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Zhu, Jianhui; Chen, Zhengwei; Zhang, Jie et al. (2018) Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS. J Proteome Res :
An, Mingrui; Zhu, Jianhui; Wu, Jing et al. (2018) Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC-1 Cells. J Proteome Res 17:1690-1699
An, Mingrui; Wu, Jing; Zhu, Jianhui et al. (2018) Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum. J Proteome Res 17:3599-3605
Luo, Xian; An, Mingrui; Cuneo, Kyle C et al. (2018) High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics. Anal Chem 90:8314-8319
Zhu, Jianhui; Warner, Elisa; Parikh, Neehar D et al. (2018) Glycoproteomic markers of hepatocellular carcinoma-mass spectrometry based approaches. Mass Spectrom Rev :
Huang, Yifan; Zhou, Shiyue; Zhu, Jianhui et al. (2017) LC-MS/MS isomeric profiling of permethylated N-glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients. Electrophoresis 38:2160-2167
An, Mingrui; Lohse, Ines; Tan, Zhijing et al. (2017) Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy. J Proteome Res 16:1763-1772
Tan, Zhijing; Nie, Song; McDermott, Sean P et al. (2017) Single Amino Acid Variant Profiles of Subpopulations in the MCF-7 Breast Cancer Cell Line. J Proteome Res 16:842-851
Zhu, Jianhui; Wu, Jing; Pei, Xiucong et al. (2017) Annexin A10 is a candidate marker associated with the progression of pancreatic precursor lesions to adenocarcinoma. PLoS One 12:e0175039
Yin, Haidi; Zhu, Jianhui; Wu, Jing et al. (2016) A procedure for the analysis of site-specific and structure-specific fucosylation in alpha-1-antitrypsin. Electrophoresis 37:2624-2632

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