Analysis of Haemophilus influenzae Strains Using a Novel Mass Spectrometry Approach
¹Andrew Carlone, ²Jennifer Whitmon, ²Kaneatra Simmon, ²Yulanda Williamson, ²Adrian Woolfitt, ²Jon Rees, ²Ellie Bromberek, ²Jackie Sampson, ²Hercules Moura, ²Eddie Ades
¹Emory University, Atlanta, GA
²Centers for Disease Control and Prevention, Atlanta, GA

Introduction

Haemophilus influenzae (Hi) is a gram negative, opportunistic bacteria which is the causative agent of various human diseases such as pneumonia, meningitis, bacteremia, conjunctivitis, and sinusitis.



Strains are encased by a polysaccharide capsule and designated serotype A-F based on the unique structural characteristics of the polysaccharide.

Although the development of a vaccine (1988) against Hi serotype B drastically reduced the number of invasive disease cases, Haemophilus, still remains the cause of thousands of deaths each year globally.

Using proteomics, (an analysis of the entire expressed protein profile of an organism) the immunoreactivity of Hi may be more fully characterized.

Mass spectrometry is a powerful analytical tool used to quantitate and identify proteins.

MALDI-TOF (Matrix Assisted Laser Desorption/ Ionization – Time of Flight) allows for efficient cell fingerprinting and, thus, provides a valuable tool for biomarker discovery.



Electro spray Ionization Mass Spectrometry (ESI-MS) is a technique which couples liquid chromatography with mass spectrometry to provide a sensitive method to identify proteins.

ESI – MS is the preferred method of ionization for protein analysis.

Because separating membrane and large hydrophobic proteins is difficult using traditional PAGE (1 and2 dimensional gel) methods, a novel technique preparing an ‘enriched membrane fraction’ was utilized.



This novel method omits the need to separate proteins by gel electrophoresis before digesting and analyzing with mass spectrometry.

Objectives

Determine whether expressed protein profiles of Haemophilus influenzae strains exhibit similarities and differences using mass spectrometry.

Identify immunoreactive proteins of H. influenzae using a novel proteomic technique and mass spectrometry.

Methods and Materials

All Hi strains were obtained from ATCC.

Four Part Procedure Outline for H. influenzae Analysis



EMF Isolation and Protein Identification



Immunoprecipitation to Isolate Immunoreactive EMF Proteins

Results

1-Dimentional Gel Showing Whole-Cell Protein Profiles



Whole-Cell MALDI Results Highlighting Differences



Dendrogram of Whole-Cell MALDI Results Indicating H. influenzae Serotype Proteomic Similarities



Truncated List of Immunoprecipitated Proteins

Conclusions and Future Studies

Whole-cell fingerprinting demonstrated both common and unique proteins

Coupling immunoprecipitation with ESI-MS allowed for identification of immunoreactive membrane proteins.

Comparative analysis of unique immunoreactive proteins is ongoing.

Newly identified proteins will be evaluated as potential vaccine and diagnostic candidates

Resources

Emory University SURE Program
Howard Hughes Medical Institute
CDC/CCID/NCIRD
CDC/CCEHIP/NCEH