¹⁸O‐labeling quantitative proteomics using an ion trap mass spectrometer

Abstract

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.

Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers

“…This mode of operation has been used for numerous quantitative proteomics studies [25][26][27][28]. Although the linear ion trap is unlikely to replace the triple quadrupole instrument (using SRM/MRM) for MS-based quantitation in the clinical setting, there are advantages of using the ion trap for early stage testing of candidate biomarkers.…”

“…Triple quadrupole instruments, already entrenched in clinical laboratories for measuring small molecules, have consistently provided measurements with high precision (CVs below 5%), a good linear response range (>10 3 ), and high sensitivity of detection (less than 1 ng/mL). Linear ion trap instruments, far more common in proteomics laboratories involved with biomarker discovery, have only recently been tested in this setting [24] but are nonetheless widely used for quantification [25][26][27][28]. To determine the performance characteristics of our magnetic beadbased capture system coupled to a linear ion trap, we assessed the linear range, limit of quantification, accuracy, and precision for our two test antigens, alpha-1-antichymotrypsin (AAC) and TNFα.…”

Rapid Validation of Mascot Search Results via Stable Isotope Labeling, Pair Picking, and Deconvolution of Fragmentation Patterns

“…etry (32,(53)(54)(55)(56)(57). We exploit the characteristic fragmentation of isotopically labeled peptides to enhance their identification, a well established principle that goes back to the period preceding the modern era of ESI and LC-MS/MS (36,37) and has since been applied effectively by a number of investigators (e.g.…”

Automated Comparative Proteomics Based on Multiplex Tandem Mass Spectrometry and Stable Isotope Labeling

“…However, the method should also be applicable to other C/N-terminal labeling techniques provided that the labeling does not cause chromatographic separation and the mass shift is small enough to allow the use of a precursor window of reasonable width. Many previously reported labeling methods such as 18 O labeling through tryptic digestion (36,37) can be used without modifying the established protocols.…”