We report both the design of a high-throughput MICROFASP (a miniaturized filter aided sample preparation) system and its use for the comprehensive proteomic analysis of single blastomeres isolated from 50-cell stage Xenopus laevis embryos (∼200 ng of yolk-free protein/blastomere). A single run of the MICROFASP system was used to process 146 of these blastomeres in parallel. Three samples failed to generate signals presumably due to membrane clogging. Two cells were lost due to operator error. Of the surviving samples, 32 were analyzed using a Q Exactive HF mass spectrometer in survey experiments (data not included). The 109 remaining blastomeres were analyzed using a capillary LC-ESI-MS/MS system coupled to an Orbitrap Fusion Lumos mass spectrometer, which identified a total of 4189 protein groups and 40,998 unique peptides. On average, 3468 ± 229 protein groups and 14,525 ± 2437 unique peptides were identified from each blastomere, which is the highest throughput and deepest proteome coverage to date of single blastomeres at this stage of development. We also compared two dissociation buffers, Newport and calcium−magnesium-free (CMFM) buffers; the two buffers generated similar numbers of protein identifications (3615 total protein IDs from use of the Newport dissociation buffer and 3671 total protein IDs from use of the CMFM buffer).
Sample preparation and separation methods determine the sensitivity and the quantification accuracy of the proteomics analysis. This article covers a comprehensive review of the recent technique development of high-throughput and high-sensitivity sample preparation and separation methods in proteomics research.
We report an integrated platform that enabled a seamlessly coupling miniaturized filter-aided sample preparation (MICROFASP) method to high-pH reversed phase (RP) or strong cation exchange (SCX) microreactors for low-loss sample preparation and fractionation of 1 μg of cell lysates prior to LC-ESI-MS/MS analysis. Due to the reduced size of the microreactor, only 5 μL of buffer volume is required to generate each fraction, which speeds both elution and lyophilization. The fraction was directly eluted into an autosampler insert vial for LC−MS analysis to reduce sample transfer steps and minimize sample loss as well as contamination. The flow-through sample generated during the loading step was also collected and analyzed. The integrated platform generated 48,890 unique peptides and 4723 protein groups from 1 μg of a K562 cell lysate using MICROFASP and C18 microreactor-based high-pH RP fractionation methods, which are comparable with the state-of-the-art result using in-StageTip sample preparation and nanoflow RPLC-based fractionation methods but with a significant reduction in cost and time. Both pH gradient elution and salt gradient elution approaches provide high reproducibility for the SCX microreactor-based fractionation method. This integrated platform has significant potential in deep proteomics analysis of mass-limited samples with reduced time and equipment requirements.
Here, we have documented a new protocol to determine D/Lamino acids by derivatizing amino acids via a chiral phosphinate. (R P )-L-Menthyl phenylphosphinate was able to bond both primary and secondary amines, as well as improve the sensitivity of analytes in MS. Eighteen pairs of amino acids were successfully labeled except for Cys which has a thiol group on the side chain, and the chirality of amino acids can be discriminated by 31 P NMR. Seventeen pairs of amino acids were separated by a C18 column within 45 min of elution, and resolution values ranged from 2.01 to 10.76. The lowest limit of detection was 10 pM acquired at parallel reaction monitoring, in which two factors collectively contributed that the ability of protonation of phosphine oxide and the sensitivity of parallel reaction monitoring. Chiral phosphine oxides might be a promising tool in future chiral metabolomics.
Tweetable abstract Multidimensional separation methods with improved sensitivity and peak capacity and throughput allow in-depth proteome profiling of low-microgram samples.
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