Middle-down proteomics: the pursuit for longer peptides

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Middle-down proteomics (MDP) offers enhanced sequence coverage and PTM localization for proteoforms.
• Current MDP applications are limited in complex biological samples like cell lysates and biofluids.
• Understanding proteoforms and PTM networks is vital for biological and disease research.

Purpose of the Study:

• To review the potential and challenges of applying MDP to complex biological samples.
• To explore advancements in sample preparation, chromatography, mass spectrometry, and bioinformatics for MDP.
• To identify key areas for workflow optimization to drive MDP adoption.

Main Methods:

• Review of sample preparation techniques including lysis, precipitation, and alternative proteases (GluC, thermolysin).
• Discussion of advanced chromatographic methods, ion mobility (FAIMS, TIMS), and fragmentation techniques (ETD, EThcD).
• Analysis of bioinformatic challenges including missed cleavages and protease specificity.

Main Results:

• In-silico analyses highlight peptide length and charge distribution as limitations for current enzymes.
• Experimental challenges include peptide solubility, ionization efficiency, and bioinformatic complexity.
• MDP has the potential to uncover previously undetected proteoforms and PTM-rich regions.

Conclusions:

• Optimizing enzyme selection, LC-MS parameters, peptide ionization, ion mobility, fragmentation, and algorithms is crucial for high-throughput MDP.
• Addressing current limitations will enable deeper proteomic insights and advance biological research.
• MDP is poised to reveal the 'dark proteome' with further workflow development.