Emerging methods in proteomics: top-down protein characterization by multistage tandem mass spectrometry

Abstract

"Top-down" mass spectrometry methods have emerged as an attractive alternative to conventional "bottom-up" approaches for the comprehensive characterization of co- and post-translational protein modifications. Here we present a brief overview of current strategies employed for top-down protein characterization and discuss the key technical challenges and solutions associated with their implementation on a range of mass spectrometry instrument platforms. For more specific details regarding the individual strategies described herein, interested readers are referred to the references cited at the end of this article.

The potential of mass spectrometry to study iron-containing proteins used in clinical diagnosis

“…However, they also suffer from some disadvantages: (1) the identification of the protein of interest is needed before its analysis for full characterization; (2) the direct MS analysis of intact protein ions requires expensive highresolution MS instruments, such as Fourier transform ion cyclotron resonance (FTICR) instrument, not widely available; (3) the protein size (>50 kDa) and the dynamic range are still important limitations. For that reason, the development of new MS instrumentation is demanded in order to improve the measurement of very large proteins [41,42,44,45]. However, most of these methods are qualitative [46] and, for a clinical biomarker, quantitative information is mandatory in order to use the protein/peptide routinely in clinical diagnosis.…”

“…if a peptide containing a phosphorylation is lost). In any case, unknown proteins can be identified in this way by tandem MS using "bottom-up" approaches and "de novo" sequencing [41,42].…”

Importance of systems biology in engineering microbes for biofuel production

“…While traditionally completed with twodimensional electro-phoresis [ 17], high-throughput identification of proteins using mass spectrometry has become the foundation of much proteomics research for functional genomics studies. Multiple strategies now exist for the identifi-cation of proteins [ 17,18], and numerous workflows to quantify protein abundance have also been developed [ 19]. Notably, the iTRAQ technique, which uses isobaric tags to differentially label samples from up to eight conditions simultaneously [ 20], has been used to survey both protein changes [ 21] and to determine members of protein complexes [ 22].…”

Characterization of Seafood Proteins Causing Allergic Diseases

“…phosphorylated, acetylated). Some of these post-translational modified residues are difficult to be identified due to poor chromatographic resolution or detector sensitivity (Chait, 2004;Scherperel & Reid, 2007), as seen for the two peptides number 2 and 5 in Fig. 3.…”

“…The MS resolution is high enough to identify a disulfide bond, which increases the molecular weight of the protein by 2 Da after reduction. The top-down method has clearly been shown to identity even the labile post-translation modification groups (Scherperel & Reid, 2007). Two-dimensional correlation between MS/MS and MS/MS/MS was also developed to confirm the amino acid sequence in Fourier-transform ion cyclotron resonance (FTICR) instruments (Zhang & McElvain, 2000).…”