Proteomic analysis of Helicobacter pylori cellular proteins fractionated by ammonium sulfate precipitation

Abstract

Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

“…Industrial scale precipitation is possible using existing bioprocessing equipment and disposable technology. Precipitation can effectively reduce HCP, product‐related impurities, and virus . Precipitation methods have been widely used in purifying proteins from blood material, ascites fluid, and egg white in combination with column chromatography .…”

“…To continuously improve the speed and ease of processing and minimize cost while maintaining the level of purity and quality of the product, it is essential to develop an alternative purification method which can replace one chromatography step and overcome the potential capacity constraints on a facility. Precipitation‐based purification technologies have the potential to remove impurities at higher protein concentration, allowing higher antibody titer cell culture broth (CB) to be processed with the same equipment . By replacing a polishing chromatography step with a precipitation technology, it is possible to alleviate some facility fit constraints and could reduce processing time under more competitive cost.…”

“…Precipitation-based purification technologies have the potential to remove impurities at higher protein concentration, allowing higher antibody titer cell culture broth (CB) to be processed with the same equipment. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] By replacing a polishing chromatography step with a precipitation technology, it is possible to alleviate some facility fit constraints and could reduce processing time under more competitive cost.…”

“…Industrial precipitation methods include ethanol precipitation, 17,18 ammonium sulfate, 9,11,13,15 Cohn fractionation, 12 or polyethylene glycol precipitation 5,10,16 and caprylic acid (CA) precipitation. 6,7,12,14,19 CA is an eight-carbon saturated fatty acid which is found naturally in the milk of various mammals, and as a minor constituent of coconut oil and palm kernel oil.…”

Separomics applied to the proteomics and peptidomics of low-abundance proteins: choice of methods and challenges - a review

“…Plant extracts obtained in the absence of this compound presented high noise/signal relationship in the spectrometry analyses and protein agglomeration was observed. Recently, Park et al (2008) stressed the importance of this procedure to remove interfering molecules. Nevertheless, caution should be taken, as the presence of salt may interfere with electrophoretic properties and the MS procedure.…”