Identification and Characterization of Sulfolobus solfataricus P2 Proteome Using Multidimensional Liquid Phase Protein Separations

Abstract

We have identified and characterized the proteome of Sulfolobus solfataricus P2 using multidimensional liquid phase protein separations. Multidimensional liquid phase chromatography was performed using ion exchange chromatography in the first dimension, followed by reverse-phase chromatography using 500 microm i.d. poly(styrene-divinylbenzene) monoliths in the second dimension to separate soluble protein lysates from S. solfataricus. The 2DLC protein separations from S. solfataricus protein lysates enabled the generation of a 2D liquid phase map analogous to the traditional 2DE map. Following separation of the proteins in the second dimension, fractions were collected, digested in solution using trypsin and analyzed using mass spectrometry. These approaches offer significant reductions in labor intensity and the overall time taken to analyze the proteome in comparison to 2DE, taking advantage of automation and fraction collection associated with this approach. Furthermore, following proteomic analysis using 2DLC, the data obtained was compared to previous 2DE and shotgun proteomic studies of a soluble protein lysate from S. solfataricus. In comparison to 2DE, the results show an overall increase in proteome coverage. Moreover, 2DLC showed increased coverage of a number of protein subsets including acidic, basic, low abundance and small molecular weight proteins in comparison to 2DE. In comparison to shotgun studies, an increase in proteome coverage was also observed. Furthermore, 187 unique proteins were identified using 2DLC, demonstrating this methodology as an alternative approach for proteomic studies or in combination with 2DE and shotgun workflows for global proteomics.

Proteome analysis of Thermococcus onnurineus NA1 reveals the expression of hydrogen gene cluster under carboxydotrophic growth

“…The total number of proteins identified from two independent LC-MS/MS analyses (1395) corresponded to 70.6% of the 1976 predicted genes of this microorganism (Table 1). To our knowledge, this protein identification rate (PIR) surpassed those reported for other Archaea, including T. gammatolerans (44%) [5], Pyrococcus furiosus (44%) [20], Haloferax volcanii (32%) [21], and Sulfolobus solfataricus (23%) [22]. The identified proteins had an average molecular weight (Mr) of 35 kDa (range 10-50 kDa), and included a magnesium and cobalt transporter (ton0668, the smallest Mr, 4.8 kDa) and a hypothetical protein (ton1824, the highest Mr, 211 kDa).…”

A cool tool for hot and sour Archaea: Proteomics of Sulfolobus solfataricus

“…Recently, using one of the more current techniques called multidimensional liquid‐phase protein separations for S. solfataricus , Assiddiq et al were able to detect up to 688 unique proteins (covering 23.1% of the theoretical proteome of S. solfataricus ) from 389 fractions collected from 2DLC. This technique applies a combination of ion exchange chromatography (first dimension) and then RP chromatography (second dimension).…”

“…It offered an increase of both proteome coverage compared to 2DE and numbers of acidic or basic proteins, which are normally undetected using traditional 2DE [30]. Furthermore, one of the benefits of this technique is the reduced labor time (11 days compared to approximately 17 days for 2DE) [30]. A new technique named precursor acquisition independent of ion count (based on LC-MS/MS) was first applied to analyze the phosphoproteome of S. solfataricus, offering exciting information on changes in response to the offered carbon source (glucose versus tryptone).…”

Characterization of a Functional DnaG-Type Primase in Archaea: Implications for a Dual-Primase System

“…43 Therefore, individual roles for each primase probably exist in Sulfolobus. Analysis of a global single base-pair map of RNA transcripts and operons in Sso reveals the relative amount of each gene that is transcribed, normalized to its length in bases.…”