プロテオミクス of Hiro's Lab

Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with genomics, the study of the genes. The word "proteome" is a blend of "protein" and "genome", and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student. The proteome is the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses, that a cell or organism undergoes. Link to Wikipedia

In the past, two-dimentional gel (2D gel) electrophoresis based methods were generally used for proteomics. However, recently, a novel approach so-called shotgun proteomics becoming popular.

High-throughput and large-scale proteomics approach, which was enabled by advances in liquid chromatography-mass spectrometry (LC-MS)-based techniques together with complete genome sequencing. Typically, in shotgun proteomics, trypsinized peptides derived from complex protein samples are analyzed by LC-MS. By shotgun proteomics thousands of proteins can be simultaneously identified from complex samples such as cell lysates.

In addition, shotgun proteomics has the potential to monitor all sorts of post-translational modifications (PTMs). However, the current proteomics technology based on large-scale analysis of PTMs requires several improvements. The number of peptides with a single PTM comprises a very small fraction of the total number of peptides produced; therefore, very few peptides with PTMs are identified. One of the key requirements for successful PTM-oriented proteomics (modificomics) is the establishment of efficient enrichment methods for posttranslationally modified peptides or proteins.

Posttranslational modifications (PTMs) represent the most common mechanism by which protein functions can be altered. Cellular signaling networks usually utilize PTMs to transmit signals. Therefore, to completely understand the molecular mechanisms of the signaling pathways and to isolate the signaling factors, it is necessary to monitor the PTM status of the proteins during signal transduction. Proteomics is one of the best available tools for studying PTMs, and it has no limitations like those encountered with forward genetics; therefore, it is well suited for the analysis of unknown signaling pathways.

Shotgun phosphoproteomics has become feasible in various organisms, including plants, owing to recent technological breakthroughs. Phosphopeptide enrichment technique, the key technology for successful shotgun phosphoproteomics, is one of the most rapidly developing techniques in the field of modificomics.

Pioneering studies on plant phosphoproteomes were restricted to subfractionated samples such as plasma membrane proteins, which contain a few hundred phosphoproteins. Because the phosphopeptide enrichment methods were as yet undeveloped. Most of the past phosphoproteomics studies have utilized 2-step phosphopeptide purification methods, i.e., prefractionation with strong cation exchange (SCX) chromatography followed by affinity chromatography such as immobilized metal affinity chromatography (IMAC) or metal oxide chromatography (MOC). However, SCX chromatography is complicated and difficult, often resulting in sample loss.

Recently, a highly efficient single-step phosphopeptide enrichment method, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC), was developed. Application of HAMMOC to plant materials made it possible to identify thousands of phosphopeptides from unfractionated plant cell lysates efficiently.

Modified version of MOC where metal oxide beads are dynamically coated with aliphatic hydroxy acids, such as lactic acid. This dynamic coating reduces the interaction with acidic peptides, but does not affect the affinity for phosphopeptides.

Affinity chromatography to enrich phosphopeptides, utilizing metal oxides such as titania, zirconia, or alumina for phosphate interaction in a similar way to IMAC.

Affinity chromatography to enrich phosphopeptides. This utilizes metal cations such as Fe3+, Ga3+ Al3+ Co2+ and Zr4+ to coordinate phosphate groups.

Chromatographic method often used to separate digested peptides. Since phosphate groups on phosphopeptides acquire negative charge at acidic pH (<2.7), phosphopeptides can be roughly separated from non-phosphopeptides using SCX. Therefore, SCX is often used prior to IMAC and MOC to reduce both the sample complexity and the contamination with non-phosphorylated acidic peptides.

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Proteomics has become a critical tool in the functional understanding of plant processes at the molecular level. Proteomics-based studies have contributed to the ever expanding array of data in modern biology, with many generating web portals and online resources. Many of these resources reflect specialist research areas with significant and novel information that is not currently captured by centralized repositories. Consequently the MASCP Proteomics Subcommittee have developed a proteomics aggregation utility called the MASCP Gator, that pulls information on-the-fly from a variety of proteomics resources.

Link to MASCP Gator

The RIPP-DB provides information on phosphopeptides which were identified from Arabidopsis and rice cells by LC-MS/MS-based shotgun phosphoproteomics approach. MS/MS spectral information for the identified phosphopeptides at PepBase (Keio University) are available. Alignment views of orthologous proteins in Arabidopsis and rice mapped with the identified phosphopeptides allow users to predict conserved phosphorylation sites in plants. Current database houses information for 5,143 and 6,919 phosphopeptides from Arabidopsis and rice, respectively.

Link to RIPP-DB