Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression

Abstract

We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis. The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests. The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components. Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30. However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

Quantitative proteomics of intracellular Porphyromonas gingivalis

“…We know, for example, that any such "within sample" interpretation will be compromised by major differences in quantitative detectability among proteins [17], depending on such factors as their true abundance, molecular weight, stability during the extraction and analytical procedures, hydrophobicity, isoelectric point and the degree to which the protein is intrinsic within the cell membrane. While purely qualitative detectability is remarkably consistent across the proteome for P. gingivalis [25], it has not been possible in the present studies to demonstrate an unequivocal and straightforward relationship between a protein's spectral count and its absolute quantity.…”

“…Some of these abundance changes have been observed in previous studies of early stages of the invasion process, prior to internalization [21,24,25]. The importance of other proteins showing pronounced abundance changes has been validated by invasion studies using knockout mutants of P. gingivalis in which the genes in question are not functional.…”

“…This decision was especially important in light of the fact that all proteomics approaches with which the authors are familiar tend to be biased in varying degrees towards the detection of proteins that are large and (or) abundant. For P. gingivalis in particular under these experimental conditions, this bias has been shown to be minimal at the level of qualitative identification [25]. However, such a bias clearly applies at the level of quantitation, where many less abundant proteins show too much scatter in the data and too few proteolytic fragments to be reliably quantified in terms of abundance ratios, using the non-label methods employed.…”

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

“…Protein Identification by 2D Gel Electrophoresis and MS. Elution fractions (1 ml) were precipitated with 0.3 volumes of acetone, and 2D gel electrophoresis was performed as described (38), with the following modifications: pH 3-10 NL (nonlinear) immobilized pH gradient (IPG) strips (Amersham Pharmacia Biosciences) were used, 1% pH 3-10 NL buffer was included in the rehydration buffer, and 12% acrylamide gels were used for the SDS͞PAGE. Gels were silver stained (39), and spots to be identified were excised by using a clean scalpel.…”

Mass Spectrometry-Based Proteomics and its Application to Studies of Porphyromonas gingivalis Invasion and Pathogenicity

“…Proteins that are differentially regulated are important components of the in vivo survival strategy of the organism. Much of the analytical and bioinformatics methodology reviewed here has been implemented in the context of this model [11][12][13], also using information derived from study of P. aeruginosa secreted virulence factors [14] and protein expression in the methanogen Methanococcus maripaludis [15,16]. …”