Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.
Multiple reaction monitoring (MRM) is a liquid chromatography−mass spectrometry (LC−MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC−MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC−MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/ mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra-and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP−mAb interaction on lipase quantification.
The green photosynthetic bacterium Chloroflexus aurantiacus, which belongs to the phylum of filamentous anoxygenic phototrophs, does not contain a cytochrome bc or bf type complex as is found in all other known groups of phototrophs. This suggests that a functional replacement exists to link the reaction center photochemistry to cyclic electron transfer as well as respiration. Earlier work identified a potential substitute of the cytochrome bc complex, now named alternative complex III (ACIII), which has been purified, identified and characterized from C. aurantiacus. ACIII functions as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain, and a related but distinct complex functions in respiratory electron flow to a terminal oxidase. In this work, we focus on elucidating the structure of the photosynthetic ACIII. We found that AC III is an integral-membrane protein complex of around 300 kDa that consists of 8 subunits of 7 different types. Among them, there are 4 metalloprotein subunits, including a 113 kDa ironsulfur cluster-containing polypeptide, a 25 kDa penta-heme c-containing subunit and two 20 kDa mono-heme c-containing subunits in the form of a homodimer. A variety of analytical techniques were employed in determining the ACIII substructure, including HPLC combined with ESI-MS, metal analysis, potentiometric titration and intensity analysis of heme-staining SDS-PAGE. A preliminary structural model of the ACIII complex is proposed based on the analytical data and chemical cross-linking in tandem with mass analysis using MALDI-TOF, as well as transmembrane and transit peptide analysis.Bacterial electron transport pathways largely fall into two major categories: the light-driven photosynthetic electron transfer chain and the aerobic or anaerobic respiratory electron transfer chain. Despite the vast differences between photo-and oxidative phosphorylations, they both couple the chemical reactions between electron donors and electron accepters to the translocation of protons across the membrane, which then drives ATP formation and other energy-dependent processes (1). As a result, the common feature of all electron transport chains is the presence of a proton pump to create the transmembrane proton gradient. In respiratory electron transfer pathways, there may be as many as three types of proton pumping protein complexes reminiscent of mitochondria, depending on * Address correspondence to: Robert E. Blankenship, Washington University in St. Louis, One Brookings Dr., CB 1137, St. Louis, . blankenship@wustl.edu. Supporting information available The identification of the cross-linked products. This supplementary material is available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 August 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript environmental factors (2). In contrast, the proton pump in all the photosynthetic electron transfer chains was u...
a b s t r a c tThe surprising lack of the cytochrome bc 1 complex in the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus suggests that a functional replacement exists to link the cyclic electron transfer chain. Earlier work identified the alternative complex III (ACIII) as a substitute of cytochrome bc 1 complex. Herein, the enzymatic activity of ACIII is studied. The results strongly support the view that the ACIII functions as menaquinol:auracyanin oxidoreductase in the C. aurantiacus electron transfer chain. Among all the substrates tested, auracyanin is the most efficient electron acceptor of ACIII, suggesting that ACIII directly transfers the electron to auracyanin instead of cytochrome c-554. The lack of sensitivity to common inhibitors of the cytochrome bc 1 complex indicates a different catalytic mechanism for the ACIII complex.
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