Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

Si Wu,Mary S Lipton,Samuel H Payne,Rui Zhao,Ljiljana Paša-Tolić,Ronald J Moore,Nikola Tolić,Da Meng,Anil Shukla,Roslyn N Brown,Li Cao,Matthew E Monroe

doi:10.1155/2013/279590

Abstract

The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.

Highlights

The periplasm of Gram-negative bacteria is a hydrated gel located between the cytoplasmic and outer membranes and is comprised of peptidoglycan, proteins, carbohydrates, and small solutes [1,2,3]
As an initial subject for analysis, we focused on the Gramnegative alphaproteobacterium, Novosphingobium aromaticivorans
The enriched intact periplasmic protein fraction was subjected to nano-LCMS/mass spectrometry (MS) using two different fragmentation methods

Summary

Introduction

The periplasm of Gram-negative bacteria is a hydrated gel located between the cytoplasmic and outer membranes and is comprised of peptidoglycan (cell wall), proteins, carbohydrates, and small solutes [1,2,3]. Localization to the periplasm and beyond often involves an N-terminal secretion signal that targets the protein for translocation across the cytoplasmic membrane via the general secretory pathway [5]. When multiple PTMs occur in a single protein, the bottom-up approach cannot accurately define proteoforms, as it does not have the ability to determine which combinations of PTMs cooccur in a single proteoform To overcome these difficulties, we employ top-down mass spectrometry (MS) to study the periplasmic proteome. The top-down approach has been successfully applied for the characterization of various protein PTMs including signal peptide identification [16]. A total of 55 proteins were confidently identified, and their PTMs were characterized including Nterminal processing (e.g., signal peptide removal), acetylation, glutathionylation, pyroglutamate modification, and disulfide bond formation

Experimental Section

Results and Discussion

Conclusions