Inverse ¹⁸O Labeling Mass Spectrometry for the Rapid Identification of Marker/Target Proteins

Abstract

Systematic analysis of proteins is essential in understanding human diseases and their clinical treatments. To achieve the rapid and unambiguous identification of marker or target proteins, a new procedure termed "inverse labeling" is proposed. With this procedure, to evaluate protein expression of a diseased or a drug-treated sample in comparison with a control sample, two converse labeling experiments are performed in parallel. The perturbed sample (by disease or by drug treatment) is labeled in one experiment, whereas the control is labeled in the second experiment. When mixed and analyzed with its unlabeled counterpart for differential comparison using mass spectrometry, a characteristic inverse labeling pattern of mass shift will be observed between the two parallel analyses for proteins that are differentially expressed. In this study, protein labeling is achieved through 18O incorporation into peptides by proteolysis performed in [18O]water. Once the peptides are identified with the characteristic inverse labeling pattern of 18O/16O ion intensity shift, MS data of peptide fingerprints or peptide sequence information can be used to search a protein database for protein identification. The methodology has been applied successfully to two model systems in this study. It permits quick focus on the signals of differentially expressed proteins. It eliminates the detection ambiguities caused by the dynamic range of detection on proteins of extreme changes in expression. It enables the detection of protein modifications responding to perturbation. This strategy can also be extended to other protein-labeling methods, such as chemical or metabolic labeling, to realize the same benefits.

18O Stable Isotope Labeling in MS-based Proteomics

“…A significant increase of the 18 O labeling rate was reported by Mirza et al [22], describing accelerated oxygen-exchange if the trypsin was immobilized in the micro-spin column. Wang et al [23] proposed inverse 18 O labeling for improved peptide/protein quantitation accuracy, particularly for peptides/ proteins exhibiting extreme abundance changes.…”

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

“…Post-biosynthesis/bioprocess labeling, in contrast to metabolic labeling, is more versatile. Chemical labeling by alkylation, esterification, or acylation [9 -11] and endoprotease catalyzed labeling with 18 O at the C-terminus of peptides [12][13][14], present examples of post-biosynthesis labeling.…”

Simultaneous quantification and identification using 18O labeling with an ion trap mass spectrometer and the analysis software application “ZoomQuant”

“…Variance increases for ratios less than one, however, ratios can be verified by reverse labeling, or replicate experiments. MALDI-TOF data for 18 O-labeling based quantification gives a comparable coefficient of variation of ~20% [10,13]. Other methods that have been developed for relative quantification using 18 O and ion trap mass spectrometers typically rely on extracted ion chromatograms and produce relative standard deviations of 25-45% [11,13].…”

“…Stable isotope labeling with 18 O is very simple, inexpensive, and requires few handling steps. Isotopic labeling can be done simultaneously with proteolysis, or decoupled from digestion by post-proteolytic labeling [8,[10][11][12]. Isotopically labeled peptide pairs have been shown to co-chromatograph, providing accurate quantitation from a single chromatographic peak [13].…”