Statistical analysis of variation in the human plasma proteome.

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.

Hydrogen production by a hyperthermophilic membrane-bound hydrogenase in water-soluble nanolipoprotein particles.

Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBHs), poor water solubility. Nanolipoprotein particles (NLPs) formed from apolipoproteins and phospholipids offer a novel means of incorporating MBHs into a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity, and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen-production devices.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Different apolipoproteins impact nanolipoprotein particle formation.

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.

Statistical analysis of the experimental variation in the proteomic characterization of human plasma by two-dimensional difference gel electrophoresis.

The complexity of human plasma presents a number of challenges to the efficient and reproducible proteomic analysis of differential expression in response to disease. Before individual variation and disease-specific protein biomarkers can be identified from human plasma, the experimental variability inherent in the protein separation and detection techniques must be quantified. We report on the variation found in two-dimensional difference gel electrophoresis (2-D DIGE) analysis of human plasma. Eight aliquots of a human plasma sample were subjected to top-6 highest abundant protein depletion and were subsequently analyzed in triplicate for a total of 24 DIGE samples on 12 gels. Spot-wise standard deviation estimates indicated that fold changes greater than 2 can be detected with a manageable number of replicates in simple ANOVA experiments with human plasma. Mixed-effects statistical modeling quantified the effect of the dyes, and segregated the spot-wise variance into components of sample preparation, gel-to-gel differences, and random error. The gel-to-gel component was found to be the largest source of variation, followed by the sample preparation step. An improved protocol for the depletion of the top-6 high-abundance proteins is suggested, which, along with the use of statistical modeling and future improvements in gel quality and image processing, can further reduce the variation and increase the efficiency of 2-D DIGE proteomic analysis of human plasma.