Quantification of intact covalently metal labeled proteins using ESI-MS/MS

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Mass spectrometry has evolved for protein quantification using stable isotope labeling.
• Lanthanide-ion-based tags are established for quantification via inductively coupled plasma-MS.

Purpose of the Study:

• To extend lanthanide-ion-based tags for quantifying intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS.
• To demonstrate the quantification of intact proteins without prior digestion.

Main Methods:

• Application of the MeCAT-iodoacetamide (IA) metal chelate tag for labeling cysteine residues on proteins.
• Separation of labeled proteins using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS.
• Fragmentation of labeled proteins using infrared multiphoton dissociation or higher-energy collision dissociation for quantification.

Main Results:

• Complete labeling of cysteine residues on large proteins was achieved with minimal excess MeCAT-IA reagent.
• Characteristic fragments were generated for quantification of intact proteins, avoiding digestion.
• Human serum albumin was successfully quantified in blood serum, with data validated by ICP-MS.

Conclusions:

• Lanthanide-based tags (MeCAT-IA) are effective for quantitative intact protein analysis via ESI-MS/MS.
• This approach simplifies protein quantification and offers inherent multiplexing potential due to the use of various lanthanides.
• The method provides a validated alternative for protein quantification in complex biological samples like serum.