Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

Abstract

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.

Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen

“…However, the putative function of Mig-14 as a transcription factor (42) suggests that at least this partially protective antigen resides in the Salmonella cytoplasm. Surface localization can be directly determined by using proteome techniques (43), but this is difficult for ex vivo samples. Surface localization can also be predicted in silico based on sequence motifs, but experimental data for various bacteria indicate that many surface-exposed antigens actually lack such motifs (1,43).…”

“…Surface localization can be directly determined by using proteome techniques (43), but this is difficult for ex vivo samples. Surface localization can also be predicted in silico based on sequence motifs, but experimental data for various bacteria indicate that many surface-exposed antigens actually lack such motifs (1,43). Taken together, potentially relevant additional antigen properties are currently difficult to analyze on a genome-wide scale and, even for individual antigens, detailed characterization might be actually more time-consuming compared to immunization experiments that yield direct protectivity data.…”

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

“…Biotinylation and isolation of P. aeruginosa surface proteins Surface biotinylation of intact P. aeruginosa was performed, as described (25). Briefly, bacteria (2 3 10 11 ) were washed in buffer A (PBS, 1 mM CaCl 2 , 0.5 mM MgCl 2 ) and resuspended in buffer B (buffer A supplemented with 1.6 mM D-biotin).…”

“…Subsequently, proteins were reduced and alkylated using 10 mM DTT and 100 mM iodoacetamide in 25 mM NH 4 HCO 3 , washed with 25 mM NH 4 HCO 3 , and dehydrated with acetonitrile. Digestion with trypsin, MALDI-TOF-mass spectrometry (MS), and protein identification by peptide mass fingerprinting were performed, as described (25).…”

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

“…Other novel proteins with a more restricted expression were also identified. Sabarth et al (2002) have applied a similar biotinylation approach to the identification of surface-membrane proteins recovered from Helicobacter pylori. Eighty-two biotinylated proteins were resolved by 2D PAGE, and a total of 18 proteins was characterized by MALDI-TOF MS.…”