Extracellular proteins as potential biomarkers in Sepsis-related cerebral injury.

Background: Sepsis can cause brain damage known as septic encephalopathy (SAE), which is linked to higher mortality and poorer outcomes. Objective clinical markers for SAE diagnosis and prognosis are lacking. This study aimed to identify biomarkers of SAE by investigating genes and extracellular proteins involved in sepsis-induced brain injury. Methods: Extracellular protein differentially expressed genes (EP-DEGs) from sepsis patients' brain tissue (GSE135838) were obtained from Gene Expression Omnibus (GEO) and evaluated by protein annotation database. The function and pathways of EP-DEGs were examined using GO and KEGG. Protein-protein interaction (PPI) networks were built and crucial EP-DEGs were screened using STRING, Cytoscape, MCODE, and Cytohubba. The diagnostic and prognostic accuracy of key EP-DEGs was assessed in 31 sepsis patients' blood samples and a rat cecal ligation and puncture (CLP)-induced sepsis model. Cognitive and spatial memory impairment was evaluated 7-11 days post-CLP using behavioral tests. Blood and cerebrospinal fluid from 26 rats (SHAM n=14, CLP n=12) were collected 6 days after CLP to analyze key EP-DEGs. Results: Thirty-one EP-DEGs from DEGs were examined. Bone marrow leukocytes, neutrophil movement, leukocyte migration, and reactions to molecules with bacterial origin were all enhanced in EP-DEGs. In comparison to the sham-operated group, sepsis rats had higher levels of MMP8 and S100A8 proteins in their venous blood (both p<0.05) and cerebrospinal fluid (p=0.0506, p<0.0001, respectively). Four important extracellular proteins, MMP8, CSF3, IL-6, and S100A8, were identified in clinical peripheral blood samples. MMP8 and S100A8 levels in the peripheral blood of sepsis patients were higher in SAE than in non-SAE. In comparison to MMP8, S100A8 had a higher area under the curve (AUC: 0.962, p<0.05) and a higher sensitivity and specificity (80% and 100%, respectively) than MMP8 (AUC: 0.790, p<0.05). High levels of S100A8 strongly correlated with 28-day mortality and the Glasgow Coma Scale (GCS) scores. Conclusion: The extracellular proteins MMP8, CSF3, IL-6, and S100A8 may be crucial in the pathophysiology of SAE. S100A8 and MMP8 are possible biomarkers for SAE's onset and progression. This research may help to clarify the pathogenesis of SAE and improve the diagnosis and prognosis of the disease.

Rapid, high-throughput method for the quantification of thyroid hormones in rat blood serum using isotope-dilution LC-MS/MS.

Aim: Numerous guideline studies required for regulatory toxicity testing now include the measurement of the thyroid hormones 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) in blood serum from rodents. A rapid, high-throughput method for the determination of the thyroid hormones T4 and T3 is reported. Materials & methods: Sample preparation is done using a 96-well microtiter plate format. Stable isotope analogs of both hormones are used as internal standards for study and quality control samples. Results & conclusion: The validated quantification levels are T3: 10 pg/ml and T4: 1 ng/ml, with CVs of <10% at the limit of quantification and up to 50*limit of quantification. The use of isotope analog internal standards eliminates the need for quantitative transfers and complete evaporations.

A binary matrix for the rapid detection and characterization of small-molecule cardiovascular drugs by MALDI-MS and MS/MS.

A mixture of α-cyano-4-hydroxycinnamic acid and 1,5-diaminonaphthalene was discovered as a novel binary matrix for the qualitative analysis of 14 small-molecule (~250-550 Da) cardiovascular drugs by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MS/MS in either positive or negative ion mode.

Limited proteolysis in porous membrane reactors containing immobilized trypsin.

Proteolysis is often a critical step in protein characterization via mass spectrometry. Compared to complete digestion, limited proteolysis gives larger peptides, and the dominant cleavage sites may identify highly accessible, flexible protein regions. This paper explores controlled proteolysis in porous nylon membranes containing immobilized trypsin. Passage of protein solutions through ∼100 µm thick membranes provides reaction residence times as short as milliseconds to limit digestion. Additionally, variation of the membrane pore size and the protease-immobilization method (electrostatic adsorption or covalent anchoring to adsorbed polymer in membrane pores) affords control over the proteolysis rate. When digesting the highly labile protein ß-casein, large membrane pores (5.0 µm) and covalent enzyme anchoring to adsorbed polymer lead to particularly long tryptic peptides. With the more trypsin-resistant proteins cytochrome c and apomyoglobin, in-membrane proteolysis with short residence times, 1.2 µm membrane pores, and trypsin electrostatically immobilized to an adsorbed polyanion cleaves the proteins after lysine residues in flexible regions. For both cytochrome c and apomyoglobin, cleavages in an interhelix region yield two particularly large peptides that cover the entire protein sequence.

Layer-by-Layer Deposition with Polymers Containing Nitrilotriacetate, A Convenient Route to Fabricate Metal- and Protein-Binding Films.

This paper describes a convenient synthesis of nitrilotriacetate (NTA)-containing polymers and subsequent layer-by-layer adsorption of these polymers on flat surfaces and in membrane pores. The resulting films form NTA-metal-ion complexes and capture 2-3 mmol of metal ions per mL of film. Moreover, these coatings bind multilayers of polyhistidine-tagged proteins through association with NTA-metal-ion complexes. Inclusion of acrylic acid repeat units in NTA-containing copolymers promotes swelling to increase protein binding in films on Au-coated wafers. Adsorption of NTA-containing films in porous nylon membranes gives materials that capture ∼46 mg of His-tagged ubiquitin per mL. However, the binding capacity decreases with the protein molecular weight. Due to the high affinity of NTA for metal ions, the modified membranes show modest leaching of Ni(2+) in binding and rinsing buffers. Adsorption of NTA-containing polymers is a simple method to create metal- and protein-binding films and may, with future enhancement of stability, facilitate development of disposable membranes that rapidly purify tagged proteins.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Immobilization of carboxymethylated polyethylenimine-metal-ion complexes in porous membranes to selectively capture his-tagged protein.

Membrane adsorbers rapidly capture tagged proteins because flow through membrane pores efficiently conveys proteins to binding sites. Effective adsorbers, however, require membrane pores coated with thin films that bind multilayers of proteins. This work employs adsorption of polyelectrolytes that chelate metal ions to create functionalized membranes that selectively capture polyhistidine-tagged (His-tagged) proteins with binding capacities equal to those of high-binding commercial beads. Adsorption of functional polyelectrolytes is simpler than previous membrane-modification strategies such as growth of polymer brushes or derivatization of adsorbed layers with chelating moieties. Sequential adsorption of protonated poly(allylamine) (PAH) and carboxymethylated branched polyethylenimine (CMPEI) leads to membranes that bind Ni(2+) and capture ∼60 mg of His-tagged ubiquitin per mL of membrane. Moreover, these membranes enable isolation of His-tagged protein from cell lysates in <15 min. The backbone amine groups in CMPEI likely increase swelling in water to double protein binding compared to films composed of PAH and the chelating polymer poly[(N,N-dicarboxymethyl)allylamine] (PDCMAA), which has a hydrocarbon backbone. Metal leaching from PAH/CMPEI- and PAH/PDCMAA-modified membranes is similar to that from GE Hitrap FF columns. Eluates with 0.5 M imidazole contain <10 ppm of Ni(2+).

Increased protein sorption in poly(acrylic acid)-containing films through incorporation of comb-like polymers and film adsorption at low pH and high ionic strength.

In principle, incorporation of comb-like block copolymers in multilayer polyelectrolyte films can both increase film thickness relative to coatings containing linear polymers and provide more swollen films for increased sorption of proteins. In the absence of added salt, alternating adsorption of 5 bilayers of protonated poly(allylamine) (PAH) and comb-like poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid) (PHEMA-g-PAA) leads to ∼2-fold thicker coatings than adsorption of PAH and linear PAA, and the difference in the thicknesses of the two coatings increases with the number of bilayers. Moreover, the (PAH/PHEMA-g-PAA)n films sorb 2- to 4-fold more protein than corresponding films prepared with linear PAA, and coatings deposited at pH 3.0 sorb more protein than coatings adsorbed at pH 5.0, 7.0, or 9.0. In fact changes in deposition pH and addition of 0.5 M NaCl to polyelectrolyte adsorption solutions alter protein sorption more dramatically than variations in the constituent polymer architecture. When deposited from 0.5 M NaCl at pH 3.0, both (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 films increase in thickness more than 400% upon adsorption of lysozyme. These films contain a high concentration of free -COOH groups, and subsequent deprotonation of these groups at neutral pH likely contributes to increased protein binding. Lysozyme sorption stabilizes these films, as without lysozyme films deposited at pH 3.0 from 0.5 M NaCl desorb at neutral pH. Films deposited at pH 9.0 from 0.5 M NaCl are more stable and also bind large amounts of lysozyme. The high binding capacities of these films make them attractive for potential applications in protein isolation or immobilization of enzymes.

Limited proteolysis via millisecond digestions in protease-modified membranes.

Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 µm) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.

Formation of high-capacity protein-adsorbing membranes through simple adsorption of poly(acrylic acid)-containing films at low pH.

Layer-by-layer polyelectrolyte adsorption is a simple, convenient method for introducing ion-exchange sites in porous membranes. This study demonstrates that adsorption of poly(acrylic acid) (PAA)-containing films at pH 3 rather than pH 5 increases the protein-binding capacity of such polyelectrolyte-modified membranes 3-6-fold. The low adsorption pH generates a high density of -COOH groups that function as either ion-exchange sites or points for covalent immobilization of metal-ion complexes that selectively bind tagged proteins. When functionalized with nitrilotriacetate (NTA)-Ni(2+) complexes, membranes containing PAA/polyethylenimine (PEI)/PAA films bind 93 mg of histidine(6)-tagged (His-tagged) ubiquitin per cm(3) of membrane. Additionally these membranes isolate His-tagged COP9 signalosome complex subunit 8 from cell extracts and show >90% recovery of His-tagged ubiquitin. Although modification with polyelectrolyte films occurs by simply passing polyelectrolyte solutions through the membrane for as little as 5 min, with low-pH deposition the protein binding capacities of such membranes are as high as for membranes modified with polymer brushes and 2-3-fold higher than for commercially available immobilized metal affinity chromatography (IMAC) resins. Moreover, the buffer permeabilities of polyelectrolyte-modified membranes that bind His-tagged protein are ~30% of the corresponding permeabilities of unmodified membranes, so protein capture can occur rapidly with low-pressure drops. Even at a solution linear velocity of 570 cm/h, membranes modified with PAA/PEI/PAA exhibit a lysozyme dynamic binding capacity (capacity at 10% breakthrough) of ~40 mg/cm(3). Preliminary studies suggest that these membranes are stable under depyrogenation conditions (1 M NaOH).

Bifunctional polymer brushes for low-bias enrichment of mono- and multi-phosphorylated peptides prior to mass spectrometry analysis.

Polymer brushes orthogonally derivatized with oxotitanium and nitrilotriacetate-Fe(III) groups enrich both mono- and multi-phosphorylated peptides for mass spectrometry analysis.

The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways.

Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 A resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.