Ion Mobility-Assisted Free Radical Initiated Peptide Sequencing
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Nicholas Borotto
PROVIDER: MSV000095935 | MassIVE |
REPOSITORIES: MassIVE
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Project description:Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Further, a trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated with data-dependant acquisition mode has allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating ion flux in TIMS affects overall performance of proteome profiling. However, the effect of TIMS settings for analyzing low-input samples has been less investigated. Thus, we sought to optimize conditions of TIMS in regards of ion accumulation/ramp times and ion mobility range for low-input samples. We observed that ion accumulation times of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 Vs cm-2 resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We applied these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1,116, and 1,651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from low number of cells was sufficient to delineate several essential metabolic pathways and T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that these parameters could be applied to label-free analysis of single cells obtained from clinically relevant samples.
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Project description:Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry (MS)-based proteomics, because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation – serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility separated (TIMS) quadrupole time-of-flight (TOF) mass spectrometer. This requires alignment of the ion mobility separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared to DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and ion mobility plane. This Automatic Isolation Design procedure is implemented in the freely available py_diAID package. In combination with in-depth project-specific proteomics libraries and the Evosep LC system, we reproducibly identified over 7,700 proteins in a human cancer cell line in 44 minutes with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 minutes LC gradients), we consistently quantified more than 6,000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor (EGF) in triplicate 21 minutes runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.
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