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Mass Spectrometry

Explore Poochon’s Proteomics Solutions

With accumulated experience and expertise in the biological application of mass spectrometry, Poochon’s scientists developed a variety of mass spectrometry-based protocols and workflows for analysis of proteins and proteomes from any biological sample to support different applications. Three fundamental platforms were developed by our scientists.

PROMICFINDER Platform for Protein Identification and Quantification

PROMICFINDER, from Poochon, is a proteomic technology capable of identifying and measuring rapidly (high throughput), broadly (thousands of proteins simultaneously), and deeply (high-and low-abundance proteins).

It is a highly sensitive, quantitative, and reproducible proteomic tool for profiling up to 3,000 different proteins from a single protein gel band sample, one IP sample, or a single un-fractioned cell/tissue lysate sample by a 110-minute LC-MS/MS run.

  • Post-translational modifications can be simultaneously identified. PTMs such as phosphorylation (S/T/Y), acetylation (K), ubiquitination (K), and O-linked/N-linked glycans can be identified from both IP and lysate samples. For specific target PTMs, the enrichment of target proteins, for example by IP, is generally required.
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Proteins, the working molecules of a cell, serve a variety of functions within cells. Some are involved in structural support and movement, or in enzymatic activity, while others interact with the outside world. The set of proteins expressed in a particular cell is named its “proteome”, which determines the cell’s health and function. The study of proteins has fundamental relevance, from the basic understanding of normal cell function, such as cell differentiation, growth, and division, to informing radically new approaches for treating disease. The study of the proteome, termed “proteomics”, is a large-scale analysis of all proteins in a biological system during specific biological events. Today, mass spectrometry is the central technology employed in the field of proteomics, enabling the analysis of post-translational modifications, protein interactions, and the complete protein inventory of biological systems, and serving as an important tool in structural biology.

Mass spectrometry analysis of proteins measures the mass-to-charge ratio of ions to identify and quantify molecules in simple and complex mixtures. The development of macromolecule ionization methods, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), enabled the study of protein structure by mass spectrometry.  Ionization also allowed scientists to obtain protein mass “fingerprints” that are matched to proteins and peptides in databases to help identity unknown targets. New isotopic tagging methods led to the quantitation of target proteins both in relative and absolute quantities. The development of high-throughput and quantitative mass spectrometry proteomics workflows within the last two decades allows to quantitatively identify more than 10,000 different proteins in human cell lines within a couple of days expanding the scope of what we know about protein structure, function, modification, and global protein dynamics.

Mass spectrometry is the most powerful tool for proteomics to assess the relative abundance of proteins among biological samples. Numerous methodologies now support relative quantification measurements, providing a routine means to analyze protein expression patterns and post-translational modification states as a function of biological perturbation. Label-free quantification, SILAC labeling based quantification, and TMT isobaric multiplex (up to 18 plex) labeling quantification is available.

The most cost-efficient methods for relative quantification through mass spectrometry are isobaric mass tags. In isobaric labeling-based quantification, each sample is derivatized with a different isotopic variant of an isobaric mass tag from a set, and then the samples are pooled and analyzed simultaneously in MS. Since the tags are isobaric, peptides labeled with isotopic variants of the tag appear as a single composite peak at the same m/z value in an MS1 scan with identical liquid chromatography (LC) retention time. The fragmentation of the modified precursor ion during MS/MS (MS2) event generates two types of product ions: (a) reporter ion peaks and (b) peptide fragment ion peaks. The quantification is accomplished by directly correlating the relative intensity of reporter ions to that of the peptide selected for MS/MS fragmentation. The fragment ion peaks observed at higher m/z are specific for peptide amino acid sequence and are used for peptide identifications, which are eventually assigned to the proteins that they represent. Since every tryptic peptide can be labeled in an isobaric labeling method, more than one peptide representing the same protein may be identified, thereby increasing the confidence in both the identification and quantification of the protein. This technology has proved to be successful in numerous experimental contexts for comparative analysis upon perturbation. The Thermo Scientific™ TMT™ Mass Tag Labeling Kits and Reagents enable multiplex relative quantitation up to eighteen different peptide samples prepared from cells or tissues.

pathway analysis

Pathway analysis (PA) is widely used to interpret large “omics” data and is based on existing biological knowledge from databases with statistical testing, mathematical analyses and computational algorithms. In principle, biological information flows from the DNA/genome to protein/proteome automatically, regulating live cell status in response to internal or external signals. The genome encoding the proteome is relatively static, while the proteome is much more dynamic allowing proteins to orchestrate all biological processes precisely, ranging from central metabolism to cell structure, maintenance, and replication. It is speculated that all biological processes (on/off) are regulated and operated by the dynamics of proteins such as modifications and changes in abundance. The regulation of different cellular functions has been categorized into a number of pathways, such as the Wnt signaling pathway and the TGF signaling pathway. In each pathway, the components are generally named according to their function, including ligands, receptors, activating regulators, inhibitory regulators, and effectors. To measure the activation strength of a pathway, the protein molecules that belong to ligands, receptors, activating regulators, or inhibitory regulators are grouped as the pathway protein ontology chain (POC), and their relative abundances (ppm) are summed. Based on the summed abundance of each POC, the activation strength or activation status of a pathway is compared between two proteome profiles. Based on these scientific assumptions Poochon developed a novel pathway tool called PROMICPATH.