Abstract
Two-dimensional gel electrophoresis coupled with liquid chromatography/mass spectrometry (2DE-LC/MS) is a classic and conventional approach to separate and identify proteins in a proteome. However, this approach is conventionally looked upon as a seemingly âlow-throughputâ technique because only one to two proteins per 2D spot are often achieved; thus, it has gradually dimmed in the field of proteomics compared to seemingly âhigh-throughputâ bottom-up non-gel approaches such as isobaric tags for relative and absolute quantification (iTRAQ), peptide tandem mass tag (TMT), stable isotope labeling of amino acids in cell culture (SILAC), and label-free. With the rapid development and application of high-sensitivity mass spectrometry in proteomics, an average of over 50 or even hundreds of proteins can be identified in every 2D gel spot in an analysis of a complex human proteome, mostly low-abundance proteins, and 2DE-LC/MS can detect the protein species, to break through the conventional concept of 2DE-LC/MS, and assist its revival in the field of proteomics. Splicing and post-translational modifications are fundamental to the proteome, and are the main factors to clarify proteoforms, which enrich the concept of the proteome. The top-down and high-throughput nature of stable isotope-labeled 2DE-LC/MS for the detection, identification, and quantification of proteoforms provides a solid methodological support for the large-scale study of human proteoforms and disease-related proteoforms to clarify mechanisms of a disease and to discover reliable biomarkers for the prediction, diagnosis, and prognostic assessment of a disease.
Highlights
Two-dimensional gel electrophoresis (2DGE) coupled with mass spectrometry (2DE-MS) is a classic proteoform separation and identification approach in the field of proteomics . [1,2,3] Here, MS commonly includes matrix-assisted laser/desorption ionization-MS peptide mass fingerprinting (MALDI-MS PMF) [4] and -tandem mass spectrometry (MALDI-MS/MS) [5], and liquid chromatography (LC)-electrospray ionization (ESI)MS/MS (LC-ESI-MS/MS; LC-MS/MS; LC/MS) [6]
Two-dimensional gel electrophoresis coupled with liquid chromatography/ mass spectrometry (2DE-LC/MS) is a classic and conventional approach to separate and identify proteins in a proteome. This approach is conventionally looked upon as a seemingly “lowthroughput” technique because only one to two proteins per 2D spot are often achieved; it has gradually dimmed in the field of proteomics compared to seemingly “high-throughput” bottom-up non-gel approaches such as isobaric tags for relative and absolute quantification, peptide tandem mass tag (TMT), stable isotope labeling of amino acids in cell culture (SILAC), and label-free
With the rapid development and application of high-sensitivity mass spectrometry in proteomics, an average of over 50 or even hundreds of proteins can be identified in every 2D gel spot in an analysis of a complex human proteome, mostly low-abundance proteins, and Two-dimensional electrophoresis 2DGE (2DE)-LC/MS can detect the protein species, to break through the conventional concept of 2DELC/MS, and assist its revival in the field of proteomics
Summary
Two-dimensional gel electrophoresis (2DGE) coupled with mass spectrometry (2DE-MS) is a classic proteoform separation and identification approach in the field of proteomics . [1,2,3] Here, MS commonly includes matrix-assisted laser/desorption ionization-MS peptide mass fingerprinting (MALDI-MS PMF) [4] and -tandem mass spectrometry (MALDI-MS/MS) [5], and liquid chromatography (LC)-electrospray ionization (ESI)MS/MS (LC-ESI-MS/MS; LC-MS/MS; LC/MS) [6]. In order to promote non-gel methods such as 2DLC-MS/MS protocols to the field of proteomics, 2DGE and 2D DIGE were commonly claimed as being very time-consuming, labor-intense, and lowthroughput They were claimed to be inadequate to separate well the extremely high- or low-mass proteins and the extremely acidic- or basic-proteins [3], identify low-abundance proteins, and distinguish comigrated or overlapped proteins with very similar pI and Mr values in one single spot . If this concept was further refined to include independent analysis of the soluble and membrane proteomes, with full technical replications—as is done in parallel with 2DE— the 2DLC approach becomes even more low throughput and generally would not handle hydrophobic proteins very well . If this concept was further refined to include independent analysis of the soluble and membrane proteomes, with full technical replications—as is done in parallel with 2DE— the 2DLC approach becomes even more low throughput and generally would not handle hydrophobic proteins very well . [54,55]
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