Analysis of Leukocyte Extracts from the American Alligator using Gel Electrophoresis with Nano Liquid Chromatography with Mass Spectrometry

From Murray Mass Spectrometry Group

TP 319 - Presented at the 55th ASMS Conference on Mass Spectrometry, June 3 - 7, 2007, Indianapolis, Indiana

Analysis of Leukocyte Extracts from the American Alligator using Gel Electrophoresis with Nano Liquid Chromatography with Mass Spectrometry (PDF)

Lancia N.F. Darville,^1  Mark E. Merchant ² & Kermit K. Murray ¹

^1 Louisiana State University, Baton Rouge, Louisiana and ²McNeese State University, Lake Charles, Louisiana

Analysis of Leukocyte Extracts from the American Alligator using Gel Electrophoresis with Nano Liquid Chromatography with Mass Spectrometry

Introduction

American Alligator

Goal

Study proteins of immunological significance from the american alligator.

Background

• Why study alligators?

- Innate immune system

• Why study leukocytes?

- Antibacterial, antifungal, antiviral

- Cationic antimicrobial peptides

• Lipopolysacharide (LPS)

- Endotoxin that causes immune response

• Why study serum?

- Good source of disease biomarkers

Approach

• 1D-PAGE

- Separate proteins in leukocytes

- Enzymatic digestion using trypsin

- Analyze using nano LC ESI-MS/MS

- ID peptides with MASCOT

- MSDB & NCBInr database

• 2D-PAGE

- Separate proteins in serum

Experimental

Gels

Gel separation

• Bio-Rad Mini-PROTEAN II
• 1D-PAGE @ 100 V
• 2D-PAGE@ 200 V

LC Separation

HPLC separation

• Dionex Ultimate 3000 Dual Gradient LC
• LC with C₁₈ column using gradient elution

Analysis

QSTAR MS

• ABI QSTAR XL with ESI source
• 337 nm UV laser (oMALDI)
• Full MS scan from 320-1500 m/z
• Selected MS/MS scan <1500 m/z

Results

Separation of alligator leukocyte

Separation of alligator leukocyte

Mascot data

Separation of alligator serum

Separation of alligator serum

Conclusions

• 1D-PAGE of leukocyte

- Resolved six proteins 10 - 75 kDa

- LC of peptides from each protein

- MASCOT ID immunological peptides

• 1D-PAGE of serum

- Resolved four proteins 20 - 100 kDa

- 2D-PAGE of serum

- ID control & LPS injected differences

Future Work

• Optimize 2D-PAGE

- Depletion

- Affinity chromatography

• Isolate and sequence serum proteins

- Control vs. LPS injected

• Isolate antibiotic cationic peptides

Acknowledgements

• Louisiana Board of Reagents Funding
• Dr. Azeem Hasan