Total Retention Liquid Chromatography-Mass Spectrometry to Achieve Maximum Protein Sequence Coverage.

Peptide identification by liquid chromatography-mass spectrometry (LC-MS) requires retention and elution of peptides from the LC column. Although medium and hydrophobic peptides are readily retained by the C18 columns that are commonly used in proteomics, short and hydrophilic peptides are not retained nor measured by MS due to their elution in the void volume after sample injection. These nonretained peptides can possess important post-translational modifications, such as glycosylation or phosphorylation. We describe a total retention LC-MS method that employs a reverse phase C18 column and porous graphitic carbon (PGC) column to retain both hydrophobic and hydrophilic peptides for LC-MS analysis. Our setup uses a single valve with a trapping column and two LC pumps run at low microliter/minute flow rates to deliver separate gradients to parallel capillary C18 and PGC columns. Our capillary LC system balances the need for high sensitivity with ease of implementation as compared to other 2D LC systems that use nanocolumns with multiple trapping columns and multiport valves. We demonstrate the utility of the method identifying hydrophilic peptides that went undetected when only a C18 nanocolumn was used. These missed hydrophilic peptides include tripeptides and N-glycosylated species.

Use of Integrated Metabolic Maps as a Framework for Teaching Biochemical Pathways in the Pre-clinical Medical Curriculum.

Introduction: The Larner College of Medicine has steadily transitioned to primarily active learning-based instruction. Although evaluations praise session formats, students often highlight difficulties in synthesizing preparatory materials to integrate biochemical pathways. A student/faculty collaboration led to the development of interactive metabolic maps that illustrate pathways and link to a broader framework of metabolism. Methods: A review of the session materials identified relevant biochemical pathways, and for each pathway, we created a fillable visual diagram to highlight the interactions between all substrates, enzymes, and cofactors. Implementation of the metabolic maps began for first-year medical students in fall 2022. Evaluation data included standard student session evaluations (Likert scale and qualitative comments) and a survey specific to the metabolic maps. Results: After implementing the maps, student ratings of biochemistry/metabolism session materials significantly improved (3.2 ± 1.04 to 4.3 ± 0.87, p < 0.001), and students made positive comments about their effectiveness. Most students (77.8%) used the metabolic maps to aid in studying biochemistry content for exams and found the metabolic maps important for integrating information about metabolic pathways. The median performance on metabolism-specific questions was higher, although not statistically significant (69.23 to 77.28, ns). Discussion: The implementation of integrated metabolic maps improved student satisfaction of biochemistry/metabolism session materials. Limitations include confounding factors related to student population differences and other simultaneous curriculum changes. Implementing interactive visual aids to integrate metabolism pathways and concepts is applicable to any medical curriculum, and other longitudinal topics may benefit from this type of curricular framework. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-024-02073-1.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3µM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

The most infectious prion protein particles.

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.

Fractionation of prion protein aggregates by asymmetrical flow field-flow fractionation.

Achieving the successful separation and analysis of amyloid and other large protein aggregates can be a difficult proposition. Field-flow fractionation (FFF) is a flow-based separation method like chromatography; however, FFF is capable of high-resolution separations in the absence of a stationary matrix. Thus, FFF is a relatively gentle technique and is well suited to the task of separating large macromolecules and macromolecular complexes. Flow field-flow fractionation (FlFFF), one of the techniques in the FFF family, has been used to successfully fractionate a wide size range of prion protein aggregates, allowing their subsequent characterization by several biophysical and biochemical methods. The ability to easily adjust the strength of the field used during separation means that FlFFF could be applied to particles ranging from 1 nm to nearly 100 mum in size. This flexibility, coupled with the ability to produce fast, high-resolution separations, makes FFF a potentially valuable tool in the field of amyloid research.

Carbohydrate moieties on the procofactor factor V, but not the derived cofactor factor Va, regulate its inactivation by activated protein C.

Factor V (FV) is a single-chain plasma protein containing 13-25% carbohydrate by mass. Studies were done to determine if these carbohydrate moieties altered the activated protein C (APC)-catalyzed cleavage and inactivation of both FV and the cofactor which results from its activation by alpha-thrombin, factor Va(IIa) (FVa(IIa)). Treatment of purified FV with N-glycanase and neuraminidase under nonprotein-denaturing conditions removed approximately 20-30% of the carbohydrate from the heavy chain region of the molecule. When glycosidase-treated FV was analyzed in an aPTT (activated partial thromboplastin time)-based APC sensitivity assay, the APC sensitivity ratio (APC-SR) increased from 2.34 to 3.33. In contrast, when glycosidase-treated FV was activated with alpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produced no substantial increase in the APC-SR. Additional functional analyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components indicated that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and were inactivated at identical rates. Analyses of the APC-catalyzed cleavage of glycosidase-treated FV at Arg(306), the initial cleavage site, revealed a 10-fold rate increase when compared to untreated FV. In contrast, and consistent with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-identical rates of APC-catalyzed cleavage at both the Arg(506) and Arg(306)sites. These combined results indicate that N-linked carbohydrate moieties play a substantial role in the APC-catalyzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) cleavage sites of FV and FVa(IIa) are distinct substrates for APC.

Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor: characterization of purified platelet-derived factor V/Va.

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from approximately 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser(692) catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr(402) with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser(692) remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr(1543) rather than Arg(1545) accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.