Evaluation of the reliability of MS1-based approach to profile naturally occurring peptides with clinical relevance in urine samples.

RATIONALE: The profiling of natural urinary peptides is a valuable indicator of kidney condition. While front-end separation limits the speed of peptidomic profiling, MS1-based results suffer from limited peptide coverage and specificity. Clinical studies on chronic kidney disease require an effective strategy to balance the trade-off between identification depth and throughput. METHODS: CKD273, a urinary proteome classifier associated with chronic kidney disease, in samples from diabetic nephropathy patients was profiled in parallel using capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography with mass spectrometry (LC-MS), and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Through cross-comparison of results from MS1 of unfractionated peptides and elution-time-resolved MS1 as well as MS/MS in LC- and CE-MS approaches, we evaluated the contribution of false-positive identification to MS1-based identification and quantitation, and analyzed the benefit of front-end separation in terms of accuracy and efficiency. RESULTS: In LC- and CE-MS, although MS1 data resulted in higher number of identifications than MS/MS, elution-time-dependent analysis revealed extensive interference by non-CKD273 peptides, which would contribute up to 50% to quantitation if they are not separated from genuine CKD273 peptides. In the absence of separation, MS1 data resulted in lower numbers of identifications and abundance pattern that significantly deviated from those by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or capillary electrophoresis with tandem mass spectrometry (CE-MS/MS). CE showed higher identification efficiency even when less sample was used or achieved faster separation. CONCLUSIONS: To ensure the reliability of MS1-based urinary peptide profiling, front-end separation should not be omitted, and elution time should be used in addition to intact mass for identification. Including MS/MS in data acquisition does not compromise the speed or identification number, while benefiting data reliability by providing real-time sequence verification.

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Self-assembly of silver(i)-based high-energy metal-organic frameworks (HE-MOFs) at ambient temperature and pressure: synthesis, structure and superior explosive performance.

Two new high-energy metal-organic frameworks (HE-MOFs), {Ag2(DNMAF)(H2O)2}n (1) and {Ag2(DNMAF)}n (2) were prepared using potassium 4,4'-bis(dinitromethyl)-3,3'-azofurazanate (K2DNMAF) in a self-assembly strategy. Compound 1 exhibits a 3D HE-MOF structure with coordinated water molecules. Compound 2 exhibits compact solvent-free 3D HE-MOFs. Both compounds show good thermostability (decomposition temperature (Td) of 211 and 218 °C) and superior detonation velocities (D) of 9673 m s-1 and 10 242 m s-1, detonation pressures (P) of 50.01 GPa and 58.30 GPa, and heat of detonation (Q) of 1.95 kcal g-1 and 2.19 kcal g-1, respectively, which are even higher than those of RDX and HMX.