discovering-protein-interactions-characterizing-protein-function

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

MS BIoworks LLC

A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer. Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures. Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance. The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites. The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed. This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap. This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion. It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database.

Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein.

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.

Gas-phase reactions of multiply protonated polypeptides and metal containing anions represent a new methodology for manipulating the cationizing agent composition of polypeptides. This approach affords greater flexibility in forming metal containing ions than commonly used methods, such as electrospray ionization of a metal salt/peptide mixture and matrix-assisted laser desorption. Here, the effects of properties of the polypeptide and anionic reactant on the nature of the reaction products are investigated. For a given metal, the identity of the ligand in the metal containing anion is the dominant factor in determining product distributions. For a given polypeptide ion, the difference between the metal ion affinity and the proton affinity of the negatively charged ligand in the anionic reactant is of predictive value in anticipating the relative contributions of proton transfer and metal ion transfer. Furthermore, the binding strength of the ligand anion to charge sites in the polypeptide correlates with the extent of observed cluster ion formation. Polypeptide composition, sequence, and charge state can also play a notable role in determining the distribution of products. In addition to their usefulness in gas-phase ion synthesis strategies, the reactions of protonated polypeptides and metal containing anions represent an example of a gas-phase ion/ion reaction that is sensitive to polypeptide structure. These observations are noteworthy in that they allude to the possibility of obtaining information, without requiring fragmentation of the peptide backbone, about ion structure as well as the relative ion affinities associated with the reactants.

Gas-phase ion/ion reactions of multiply protonated polypeptides with metal containing anions.

Multiply protonated human hemoglobin alpha-chain species, ranging from [M + 4H]4+ to [M + 20H]20+, have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D75/M76, also noted previously in beam-type collisional activation studies, was observed for the [M + 8H]8+ to [M + 11H]11+ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D75/M76 channel. The [M + 12H]12+-[M + 16H]16+ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M + 4H]4+ ion, all charge states showed cleavage at the L113/P114 bond. This cleavage proved to be the most prominent dissociation for charge states [M + 14H]14+ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach.

Ion trap collision-induced dissociation of human hemoglobin alpha-chain cations.

Preimplantation factor (PIF) is a novel embryo-secreted immunomodulatory peptide. Its synthetic analog (sPIF) modulates maternal immunity without suppression. There is an urgent need to develop agents that could prevent the development of type 1 diabetes mellitus (TIDM). Herein, we examine sPIF's preventive effect on TIDM development by using acute adoptive-transfer (ATDM) and spontaneously developing (SDM) in non-obese diabetic (NOD) murine models. Diabetes was evaluated by urinary and plasma glucose, intraperitoneal glucose tolerance test (IPGTT), pancreatic islets insulin staining by immunohistochemistry and by pancreatic proteome evaluation using mass spectrometry, followed by signal pathway analysis. Continuous administration of sPIF for 4-weeks prevents diabetes development in ATDM model in >90% of recipients demonstrated by normal IPGTT, preserved islets architecture, number, and insulin staining. (P < 0.01). sPIF effect was specific; its protective effects are not replicated by scrambled PIF (χ(2) = 0.009) control. sPIF led also to increased circulating Th2 and Th1 cytokines. In SDM model, 4-week continuous sPIF administration prevented onset of diabetes for 21 weeks post-therapy (P < 0.01). Low-dose sPIF administration for 16 weeks prevented diabetes development up to 14 weeks post-therapy, evidenced by preserved islets architecture and insulin staining. In SDM model, pancreatic proteome pathway analysis demonstrated that sPIF regulates protein traffic, prevents protein misfolding and aggregation, and reduces oxidative stress and islets apoptosis, leading to preserved insulin staining. sPIF further increased insulin receptor expression and reduced actin and tubulin proteins, thereby blocking neutrophil invasion and inflammation. Exocrine pancreatic function was also preserved. sPIF administration results in marked prevention of spontaneous and induced adoptive-transfer diabetes suggesting its potential effectiveness in treating early-stage TIDM.

Preimplantation factor (PIF) analog prevents type I diabetes mellitus (TIDM) development by preserving pancreatic function in NOD mice.

Embryo-derived PIF modulates systemic maternal immunity without suppression. Synthetic analog (sPIF) prevents juvenile diabetes, preserves islet function, reducing oxidative stress/protein misfolding. We investigate sPIF effectiveness in controlling neuroinflammation/MS.

Preimplantation factor (PIF*) reverses neuroinflammation while promoting neural repair in EAE model.

In cancer cells, the process of epithelial-mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival. Utilizing a transforming growth factor-β-induced cell culture model of EMT, we quantitatively profiled differentially secreted proteins, by GeLC-tandem mass spectrometry. Integrating with the corresponding transcriptome, we derived an EMT-associated secretory phenotype (EASP) comprising of proteins that were differentially upregulated both at protein and mRNA levels. Four independent primary tumor-derived gene expression data sets of lung cancers were used for survival analysis by the random survival forests (RSF) method. Analysis of 97-gene EASP expression in human lung adenocarcinoma tumors revealed strong positive correlations with lymph node metastasis, advanced tumor stage and histological grade. RSF analysis built on a training set (n = 442), including age, sex and stage as variables, stratified three independent lung cancer data sets into low-, medium- and high-risk groups with significant differences in overall survival. We further refined EASP to a 20 gene signature (rEASP) based on variable importance scores from RSF analysis. Similar to EASP, rEASP predicted survival of both adenocarcinoma and squamous carcinoma patients. More importantly, it predicted survival in the early-stage cancers. These results demonstrate that integrative analysis of the critical biological process of EMT provides mechanism-based and clinically relevant biomarkers with significant prognostic value.

Epithelial-mesenchymal transition-associated secretory phenotype predicts survival in lung cancer patients.

The objective of this overview is to introduce bottom-up mass spectrometry (MS)-based proteomics approaches and strategies, widely used in other biomedical research fields, to the wound-healing research community.

Bottom-Up Mass Spectrometry-Based Proteomics as an Investigative Analytical Tool for Discovery and Quantification of Proteins in Biological Samples.

Circadian (~24hr) clocks control daily rhythms in metabolism, physiology and behavior in animals, plants and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences circadian period by regulating CLK activity and progression through the feedback loop.

Ravi Amunugama

MS BIoworks LLC

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology