The use of deuterium oxide (D2O) has greatly expanded the scope of what is possible for the measurement of protein synthesis. The greatest asset of D2O labeling is that it facilitates the measurement of synthesis rates over prolonged periods of time from single proteins through integrated tissue-based measurements. Because the ease of administration, the method is amenable for use in a variety of models and conditions. Although the method adheres to the same rules as other isotope methods, the flexibility can create conditions that are not the same as other approaches and thus requires careful execution to maintain validity and reliability. For this CORP article, we provide a history that gave rise to the method and discuss the advantages and disadvantages of the method, the critical assumptions, guidelines, and best practices based on instrumentation, models, and experimental design. The goal of this CORP article is to propagate additional use of D2O in a manner that produces reliable and valid data.
Sample preparation
for single-cell proteomics is generally performed
in a one-pot workflow with multiple dispensing and incubation steps.
These hours-long processes can be labor intensive and lead to long
sample-to-answer times. Here we report a sample preparation method
that achieves cell lysis, protein denaturation, and digestion in 1
h using commercially available high-temperature-stabilized proteases
with a single reagent dispensing step. Four different one-step reagent
compositions were evaluated, and the mixture providing the highest
proteome coverage was compared to the previously employed multistep
workflow. The one-step preparation increases proteome coverage relative
to the previous multistep workflow while minimizing labor input and
the possibility of human error. We also compared sample recovery between
previously used microfabricated glass nanowell chips and injection-molded
polypropylene chips and found the polypropylene provided improved
proteome coverage. Combined, the one-step sample preparation and the
polypropylene substrates enabled the identification of an average
of nearly 2400 proteins per cell using a standard data-dependent workflow
with Orbitrap mass spectrometers. These advances greatly simplify
sample preparation for single-cell proteomics and broaden accessibility
with no compromise in terms of proteome coverage.
Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometryPreterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.
Many of the diseases
that plague society today are driven by a
loss of protein quality. One method to quantify protein quality is
to measure the protein folding stability (PFS). Here, we present a
novel mass spectrometry (MS)-based approach for PFS measurement, iodination
protein stability assay (IPSA). IPSA quantifies the PFS by tracking
the surface-accessibility differences of tyrosine, histidine, methionine,
and cysteine under denaturing conditions. Relative to current methods,
IPSA increases protein coverage and granularity to track the PFS changes
of a protein along its sequence. To our knowledge, this study is the
first time the PFS of human serum proteins has been measured in the
context of the blood serum (in situ). We show that IPSA can quantify
the PFS differences between different transferrin iron-binding states
in near in vivo conditions. We also show that the
direction of the denaturation curve reflects the in vivo surface accessibility of the amino acid residue and reproducibly
reports a residue-specific PFS. Along with IPSA, we introduce an analysis
tool Chalf that provides a simple workflow to calculate
the residue-specific PFS. The introduction of IPSA increases the potential
to use protein structural stability as a structural quality metric
in understanding the etiology and progression of human disease. Data
is openly available at (project ID 1771).
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