Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care

Characterization of a hyperthermophilic P-type ATPase from Methanococcus jannaschii expressed in yeast.

We report on the biochemical and structural properties of a putative P-type H(+)-ATPase, MJ1226p, from the anaerobic hyperthermophilic Archaea Methanococcus jannaschii. An efficient heterologous expression system was developed in Saccharomyces cerevisiae and a four-step purification protocol, using n-dodecyl beta-d-maltoside, led to a homogeneous detergent-solubilized protein fraction with a yield of over 2 mg of protein per liter of culture. The three-dimensional structure of the purified detergent-solubilized protein obtained at 2.4 nm resolution by electron microscopy showed a dimeric organization in which the size and the shape of each monomer was compatible with the reported structures of P-type ATPases. The purified MJ1226p ATPase was inactive at 40 degrees C and was active at elevated temperature reaching high specific activity, up to 180 micromol of P(i) x min(-1) x mg(-1) at 95 degrees C. Maximum ATPase activity was observed at pH 4.2 and required up to 200 mm monovalent salts. The ATPase activity was stable for several days upon storage at 65 degrees C and was highly resistant to urea and guanidine hydrochloride. The protein formed catalytic phosphoenzyme intermediates from MgATP or P(i), a functional characteristic specific of P-type ATPases. The highly purified, homogeneous, stable, and active MJ1226p ATPase provides a new model for further structure-function studies of P-type ATPases

Patient ventilator asynchrony in critically ill adults: frequency and types

Patient ventilator asynchrony (PVA) occurs frequently, but little is known about the types and frequency of PVA. Asynchrony is associated with significant patient discomfort, distress and poor clinical outcomes (duration of mechanical ventilation, intensive care unit and hospital stay). Pressure-time and flow-time waveform data were collected on 27 ICU patients using the Noninvasive Cardiac Output monitor for up to 90 min per subject and blinded waveform analysis was performed. PVA occurred during all phases of ventilated breaths and all modes of ventilation. The most common type of PVA was Ineffective Trigger. Ineffective trigger occurs when the patient's own breath effort will not trigger a ventilator breath. The overall frequency of asynchronous breaths in the sample was 23%, however 93% of the sample experienced at least one incident of PVA during their observation period. Seventy-seven percent of subjects experienced multiple types of PVA. PVA occurs frequently in a variety of types although the majority of PVA is ineffective trigger. The study uncovered previously unidentified waveforms that may indicate that there is a greater range of PVAs than previously reported. Newly described PVA, in particular, PVA combined in one breath, may signify substantial patient distress or poor physiological circumstance that clinicians should investigate