According to Genome Sequencing Project statistics (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html), as of Feb 16, 2012, complete gene sequences have become available for 2816 viruses, 1117 prokaryotes, and 36 eukaryotes.1–2 The availability of full genome sequences has greatly facilitated biological research in many fields, and has greatly contributed to the growth of proteomics.

Proteins are important because they are the direct bio-functional molecules in the living organisms. The term “proteomics” was coined from merging “protein” and “genomics” in the 1990s.3–4 As a post-genomic discipline, proteomics encompasses efforts to identify and quantify all the proteins of a proteome, including expression, cellular localization, interactions, post-translational modifications (PTMs), and turnover as a function of time, space and cell type, thus making the full investigation of a proteome more challenging than sequencing a genome. There are possibly 100,000 protein forms encoded by the approximate 20,235 genes of the human genome,5 and determining the explicit function of each form will be a challenge.

The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis. Mass spectrometry has emerged as a core tool for large-scale protein analysis. In the past decade, there has been a rapid advance in the resolution, mass accuracy, sensitivity and scan rate of mass spectrometers used to analyze proteins. In addition, hybrid mass analyzers have been introduced recently (e.g. Linear Ion Trap-Orbitrap series6–7) which have significantly improved proteomic analysis.

“Bottom-up” protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up is performed on a mixture of proteins it is called shotgun proteomics,8–10 a name coined by the Yates lab because of its analogy to shotgun genomic sequencing.11 Shotgun proteomics provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Protein inference is accomplished by assigning peptide sequences to proteins. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. In contrast, another strategy, termed ‘top-down’ proteomics, is used to characterize intact proteins (Figure 1). The top-down approach has some potential advantages for PTM and protein isoform determination and has achieved notable success. Intact proteins have been measured up to 200 kDa,12 and a large scale study has identified more than 1,000 proteins by multi-dimensional separations from complex samples.13 However, the top-down method has significant limitations compared with shotgun proteomics due to difficulties with protein fractionation, protein ionization and fragmentation in the gas phase. By relying on the analysis of peptides, which are more easily fractionated, ionized and fragmented, shotgun proteomics can be more universally adopted for protein analysis. In fact, a hybrid of bottom-up and top-down methodologies and instrumentation has been introduced as middle-down proteomics.14 Essentially, middle-down proteomics analyzes larger peptide fragments than bottom-up proteomics, minimizing peptide redundancy between proteins. Additionally the large peptide fragments yield similar advantages as top-down proteomics, such as gaining further insight into post-translational modifications, without the analytical challenges of analyzing intact proteins. Shotgun proteomics has become a workhorse for the analysis of proteins and their modifications and will be increasingly combined with top-down methods in the future.



Figure 1

Proteomic strategies: bottom-up vs. top-down vs. middle-down. The bottom-up approach analyzes proteolytic peptides. The top-down method measures the intact proteins. The middle-down strategy analyzes larger peptides resulted from limited digestion or ...



In the past decade shotgun proteomics has been widely used by biologists for many different research experiments, advancing biological discoveries. Some applications include, but are not limited to, proteome profiling, protein quantification, protein modification, and protein-protein interaction. There have been several reviews nicely summarizing mass spectrometry history,15 protein quantification with mass spectrometry,16 its biological applications,5,17–26 and many recent advances in methodology.27–32 In this review, we try to provide a full and updated survey of shotgun proteomics, including the fundamental techniques and applications that laid the foundation along with those developed and greatly improved in the past several years.

Protein Analysis by Shotgun/Bottom-up Proteomics

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Bioactive Milk Peptides: A Prospectus

Virulence factors of Porphyromonas gingivalis.

Living organisms are capable of inducing the crystallization and deposition of a wide variety of minerals1 but the vertebrates mainly utilize the calcium phosphates in constructing their mineral phases in both normal circumstances in bone, dentin and tooth enamel and in pathological ectopic mineral deposits. The predominant form of the mineral in all situations is as carbonated apatite. However, the extent of mineralization in a particular tissue or organ is quite variable and crystallite size, crystal shape, and the packing and organization of the mineral crystals may also be variable. It is clear that the same physical chemical principles must apply to all, but it is equally clear that the organism must tightly regulate the local environment where the mineral is formed. This is an intrinsically complex problem because the mineral crystals of bone and dentin form in the extracellular matrix, external to the cells which are the ultimate regulators of the process.

Years of study of this complex set of problems has led to the general consensus that the cell-controlled processes of mineralization begin with the manufacture of an organic structure within which, or a compartment surface upon which, the mineral crystals may be initiated. The cells secrete and organize macromolecular structures which determine the ultimate character and orientations of the crystals subsequently initiated and grown, but in general, these structures do not themselves have the capacity to initiate mineralization. The incipient crystal nucleation event depends upon the interaction of the structural macromolecules with another set of secreted interactive macromolecules which locate specifically on the structural framework. These interactive molecules are doubly interactive, binding specifically to the framework on the one hand, and attracting the requisite mineral ions on the other to initiate the mineral ion clustering to a critical size that nucleates crystal growth. The biological fluids are supersaturated in mineral ion content with respect to the solubility of their biogenic mineral crystal phase, but unrestricted and uncontrolled crystal growth are not permissible in the vertebrate animal so there is a third line of macromolecular components interactive with the growing crystal surfaces that can limit growth rates, and do so selectively, controlling the crystal shape as well as size. A final component of the regulation is the availability of the crystal microions, pumped in controlled fashion from the cell into the extracellular matrix. The situation is shown schematically in Figure 1, wherein the solid, heavy arrows trace the construction of the structural matrix (a), the addition of the interactive macromolecules that activate the structural matrix (b) to bind the mineral ions (c), leading to growth of the oriented crystals (d), and finally, the addition of further macromolecules (e) to shape and delimit the crystal size to attain the mature mineralized matrix form. Note that in this paragraph, and in Figure 1 the nature of the macromolecules and mineral phase has not been specified. This is intentional to emphasize the point that in the majority of mineralizing tissues in animal systems the same overall scheme of events is likely to apply, although the structural scaffolds and mineral types can vary greatly.



Figure 1

A proposed generalized scheme for matrix-regulated mineralization reactions. Modified and reprinted with permission from Reference 280 [Veis, Reviews in Mineralogy & Geochemistry, V. 54]. Copyright 2003 Mineralogical Society of America.



The calcium and phosphate ion product (Ca × P) in the extracellular fluids (ECF) of the vertebrates are greater than the solubility products of most crystalline forms of calcium phosphate, with hydroxylapatite (HA) being the major least soluble form of calcium phosphate. Hence, HA would precipitate spontaneously if it were not for the inhibition of crystal formation by the reduction of the activity of these ions, and their sequestration, by various macromolecular components. Thus, the ECF proteins in Ca-rich milk and blood, for example, play important roles as inhibitors of apatite precipitation. The initial form of the Ca – P solid phase deposits in vitro have been shown to be disordered and exhibit a diffuse X-ray diffraction pattern, indicating that initially only a very short range ordering of calcium and phosphate ions relative to each other takes place, producing a structure called “amorphous calcium phosphate” (ACP) 2,3,4. Depending upon the conditions of temperature, pH, and ion concentrations, the solid phase may grow through a variety of forms from the ACP to (in order of decreasing solubility) brushite [CaHPO4·2H2O], whitlockite [Ca3(PO4)2 ·3H20], octacalcium phosphate [Ca8H2(PO4)6·5H2O], and hydroxyapatite [Ca10(PO4)6(OH)2]. In bone, dentin, cementum and enamel, in vivo, where the nucleation process and the final crystal composition are determined by the local environment created by the combination of the collagen or amelogenin structural matrix and particular NCP components, it is evident that the transition from ion aggregate to crystalline form is tightly regulated. A question of great importance for those interested in development of biomimetic models of bone is how the NCP might template the desired crystal forms.

The principal focus of this review, however, will be on the influence of the phosphoproteins of the matrix on the formation and growth of crystals in two systems: the small, plate-like crystals and crystal aggregates of carbonated apatite found in bone, dentin and cementum; and the large, high axial ratio rod-like crystal aggregates found in dental enamel. As pointed out many years ago5, the dentin and enamel are formed in apposition to each other and provide direct contrasts between the nature of the compartments created by the cells, the structural matrices produced, and the extracellular matrix macromolecules that control the nucleation, growth and structure of their carbonated apatite mineral phases. Here we consider these two systems separately.

/pdf/phosphorylated-proteins-and-control-over-apatite-nucleation-1tbiewjeur.pdf

Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition.

Casein phosphopeptides (CPP) stabilize calcium phosphate through the formation of casein-phosphopeptide amorphous calcium-phosphate complexes (CPP-CP). The ability of CPP-CP to reduce caries activity was investigated by use of specific-pathogen-free rats inoculated with Streptococcus sobrinus. The animals consumed a defined cariogenic diet free of dairy products. Solutions (100 microL) of the CPP-CP (0.1, 0.2, 0.5, 1.0% w/v) were applied to the animals' molar teeth twice daily. Other groups of animals received solutions containing 500 ppm F, the non-phosphorylated peptides of a casein tryptic digest (0.5% w/v), or the calcium-phosphate complex of a synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe, corresponding to the common sequence in the CPP. The CPP-CP significantly reduced caries activity in a dose-response fashion, with 1.0% CPP-CP producing 55% and 46% reductions in smooth surface and fissure caries activity, respectively, being similar to that of 500 ppm F. The anticariogenic effects of CPP-CP and fluoride were additive, since animals receiving 0.5% CPP-CP plus 500 ppm F had significantly lower caries activity than those animals receiving either CPP-CP or fluoride alone. The tryptic digest of casein with the phosphopeptides selectively removed showed no anticariogenic activity. The synthetic octapeptide-calcium phosphate complex significantly reduced caries activity, confirming that this calcium-phosphate-stabilizing portion of the casein phospho-peptides is associated with anticariogenicity. The CPP-CP did not significantly affect the level of S. sobrinus in fissure plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticariogenicity of Calcium Phosphate Complexes of Tryptic Casein Phosphopeptides in the Rat

A Selective Precipitation Purification Procedure for Multiple Phosphoseryl-Containing Peptides and Methods for Their Identification

A chromatography column containing hydroxyapatite beads was used to study the effect of different proteins on the rate of hydroxyapatite dissolution. The four phosphoproteins tested (phosvitin, alpha sl-casein, beta-casein and kappa-casein) markedly reduced the rate of hydroxyapatite dissolution. Three nonphosphorylated proteins had a relatively smaller effect. The effect of the protein in reducing the hydroxyapatite dissolution rate has been attributed to protein binding to the surface of hydroxyapatite. The reduction in dissolution rate, expressed as the change in nmol calcium released per min per nmol of phosphoprotein bound to hydroxyapatite, increased with increasing number of phosphoserine residues of the protein. The results are consistent with the proposition that phosphoproteins have a regulatory role in mineralization processes and could provide a mechanism by which dietary and salivary phosphoproteins exert an anticariogenic effect.

Phosphoprotein inhibition of hydroxyapatite dissolution.

Proteinase-adhesin complexes of Porphyromonas gingivalis wild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpA(cat), the Lys-specific proteinase Kgp(cat) and adhesin domains RgpA A1, RgpA A2, RgpA A3, Kgp A1 and Kgp A2. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpA A4, indicated that this band contained C-terminally truncated Kgp A1 (which has an identical N-terminus to RgpA A4) as well as the HagA A1* adhesin. Analysis of the Triton X-114 extracted complexes from the P. gingivalis isogenic mutants kgp (RgpA complex) and rgpA (Kgp complex) suggested that the Kgp complex consisted of Kgp(cat), Kgp A1 and Kgp A2/HagA A2 and that the RgpA complex consisted of RgpA(cat), RgpA A1, HagA A1*, RgpA A2 and RgpA A3. Each of the complexes was found to have equilibrium binding constants (K(D)) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower K(D) values for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.

/pdf/characterization-of-proteinase-adhesin-complexes-of-baw4ony66f.pdf

P.F. Riley

Papers

Anticariogenicity of Calcium Phosphate Complexes of Tryptic Casein Phosphopeptides in the Rat

A Selective Precipitation Purification Procedure for Multiple Phosphoseryl-Containing Peptides and Methods for Their Identification

Phosphoprotein inhibition of hydroxyapatite dissolution.

Characterization of proteinase-adhesin complexes of Porphyromonas gingivalis.

The analysis of multiple phosphoseryl-containing casein peptides using capillary zone electrophoresis