MS-based proteomics

FIGURE 15.1 Overview of configuration of MS and MS-based proteomic analysis. Proteins are extracted from biologic samples and fractionated by a variety of separation methods including gel separation, HPLC, and capillary electrophoresis. The common ion sources and mass analyzers used are indicated. 

FIGURE 15.2 Common protein ionization methods used for MS-based proteomics. Two common ionization technologies are currently available for protein analysis. Top ESI volatilizes and ionizes peptides and proteins in solution. Bottom MALDI uses analytes that are co-crystallized in a matrix composed of organic acid on a solid support. A pulse of ultraviolet laser evaporates the matrix and analyte into gas phase, resulting in generation of single charge ions. 

Introduces microparallel liquid chromatography (LC), ADME/PK high-throughput assay, MS-based proteomics, and the advances of capillary and nano-HPLC technology... 

Bossio, R.E. Marshall, A.G. Baseline Resolution of Isobaric Phosphorylated and Sulfated Peptides and Nucleotides by ESl-FT-lCR-MS Another Step Toward MS-Based Proteomics. Anal. Chem. 2002, 74, 1674-1679. 

In addition to the stable isotope labeling ( 0 versus 0) of proteins for quantifiable proteomic analyses as described above, chemical approaches to the protein-labeUng problem have developed in great variety. These so-called affinity tags can be used to label specific side chain groups such as sulfhydryl or amino groups, active sites for serine and cysteine hydrolases and many others. This active research area has been reviewed recently by A. Leitner and W. Lindner in a Proteomics article entitled Chemistry meets proteomics The use of chemical tagging reactions for MS-based proteomics. ... 

The most frequently used instruments of MS-based proteome analysis are as follows. 

J. Reinders, U. Lewandrowski, J. Moebius, Y. Wagner, A. Sickmann, Challenges in MS-based proteomics, Proteomics, 4 (2004) 3686. 

In proteomics experiments, neither CID/IRMPD (b- and j-series ions) nor ECD/ETD (c- and z-series ions) fragmentation, when used on its own, is capable of generating a complete set of sequence ions from which an unambiguous primary structure of a peptide may be derived (IRMPD and ECD in FTICR MS,86 CID and ETD in QJT and LIT,90 CID and ETD in QToF91). Thus database searching, which is at the heart of MS/MS-based proteomics, is vulnerable to misidentification of peptides (false positives).111 The respective characteristics of both these processes are summarized in Table 4. 

FIGURE 3 Categorization of MS-based proteomics quantitation approaches. 

MS-based proteomics and metabolomics have led to the development of a wide range of instruments focused on the mass range <5000 Da. This includes... 

FIGURE 1 Workflow for a common analysis of MS-based proteomics. The flashes represent where it is possible to apply different methods (bioinformatics and not) in order to increase the number of identification. 

The serum is aliquoted 20 pL per tube, stored at -70 °C and used only once after thawing. Several articles have discussed the importance of standardization of preanalytical factors such as patient preparation and sample collection methods for MS-based proteomics profiling (17,18,19,20). 

A new subdiscipline of bioinformatics, comparative proteogenomics, is based on the possibility of mapping sets of sequenced peptides onto the positions in the genome that code for them, i.e., integration of data from MS-based proteomics with DNA sequence data sets. Databases and software to determine if the available data are over- or underrepresented include Gene Ontology, protein domains, and pathway databases. Another application (of many) is the combination of MS-based proteomic data with information from transcriptomics to study the processes and regulation of cellular functions. 

Chen, S.L., Wu, S.L., Huang, L.J., et al. (2013) A global comparability approach for biosinular monoclonal antibodies using LC-tandem MS based proteomics. J Pharm BiomedAnal, 80,126-135. 

Given the sensitive, label-free, and multiplexed analysis that MS can provide and the automated, low-volume fluidic operations enabled by microfluidics, the combination of microfluidics with ESI-MS is expected to see increased use for chemical and biochemical analyses in the years to come. In particular, the ability of microfluidics to process minute volumes and perform sample handling operations in a high-throughput, automated fashion can extend MS-based proteomics and metabolomics studies to samples that are too small for current processes, including in-depth chemical analysis of single cells. 

MS-based proteome analysis has been significantly improved in the past decade and it allows for the detection of more types of proteins and proteins with low abundances comparedto 2D-DIGE-based proteome analysis. However, in our system, it is sufficient to compare a proteomic profile of the around 300 spots and it... 

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