Top-down proteomics: applications, recent developments and perspectives

Abstract

REVIEWNow-a-days, top-down proteomics (TDP) is a booming approach for the analysis of intact proteins and it is attaining significant interest in the field of protein biology. The term has emerged as an alternative to the well-established, bottom-up strategies for analysis of peptide fragments derived from either enzymatically or chemically digestion of intact proteins. TDP is applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein complexes and assemblies. This article delivers an overview of the methodologies in top-down mass spectrometry, mass spectrometry instrumentation and an extensive review of applications covering the venomics, biomedical research, protein biology including the analysis of protein post-translational modifications (PTMs), protein biophysics, and protein complexes. In addition, limitations of top-down proteomics, challenges and future directions of TDP are also discussed.

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…198 Thus, ECD could be applicable for TD characterization of proteins. 199 In fact, ECD was observed serendipitously for the rst time by Zubarev and Kelleher in McLafferty's research lab. 33 An interesting aspect is that ECD of multiply protonated proteins or peptides would predominantly yield c-and z-type ions, viz., c 0 and z ions (see Scheme 4), which arise from radical mediated cleavage process of the backbone N-C a bonds.…”

Characterizing the Top-down Sequencing of Protein Ions Prior to Mobility Separation in a timsTOF

“…The capacity to generate and annotate a diverse set of sequence informative ions increases in difficulty as the mass of the interrogated protein ion rises. [35][36][37] To evaluate the applicability of CIDtims to large protein ions, we i directly infused carbonic anhydrase (29 kDa) and bovine serum albumin (BSA) (66 kDa) and subjected each to CIDtims. Unlike ubiquitin, CytC, and ΒLG, however, when carbonic anhydrase is analyzed at Δ6 values of 30 V significant fragmentation is observed (Figure S8).…”

Mass Spectrometry as a Crucial Analytical Basis for Omics Sciences

“…(2) The so-called “top-down” proteomics is becoming more and more widespread [ 25 , 26 ]. The development of this approach is associated with the main disadvantage of the bottom-up method, namely, the complexity of “assembling” the initial peptide from its fragments.…”