Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides

Abstract

Liquid chromatography tandem mass spectrometry (LC–MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS3 spectra from target peptides. Skyline, a widely used software for SRM experiments, was used for chromatographic ion extraction. Phosphopeptides spiked into a BSA background yielded concentration response curves with high correlation coefficients (typically >0.9) and low coefficients of variation (≤15%) over a 200-fold concentration range. Stable isotope dilution (SID) and IRP methods were compared for quantitation of six site-specific phosphorylations in the epidermal growth factor receptor (EGFR) in epidermal growth factor-stimulated A431 cells with or without the addition of EGFR inhibitors cetuximab and gefitinib. Equivalent responses were observed with both IRP and SID methods, although analyses using the IRP method typically had higher median CVs (22–31%) than SID (10–20%). Analyses using both methods were consistent with immunoblot using site-selective antibodies. The ease of implementation and the suitability for targeted quantitative comparisons make this method suitable for broad application in protein biochemistry.

Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

“…Some internal standard peptides such as MassPREP TM (Waters) were already widely used. Other groups spiked an identical amount of standard protein into samples prior to protein digestion (22)(23)(24). There are two major normalization procedures.…”

Histone H2AX Y142 phosphorylation is a low abundance modification

“…To allow determination of relative ionization efficiency, these peptides were synthesized concatenated with a common quantification peptide. After trypsin digestion to separate the two peptides, the released phosphopeptides were fragmented (Figure 1a), and the indicated fragments were quantified in pseudo-SRM assays utilising the linear ion trap of an Orbtrap XL [15]. The H2AX Cterminal peptides were detected in both singly-and doubly-charged forms.…”

“…Nanospray was from a New Objective emitter with 10 μm tip (FS360-20-10-N-20). Pseudo-SRM was carried out in the linear ion trap of an Orbitrap XL, with a precursor isolation window of 2 m/z, an iontrap fill time of maximum 50 ms and an LTQ target ion count of 1E4 [15]. A high-resolution precursor scan was carried out in the Orbitrap (5E5 target).…”

“…In this analysis we employed a propionylation derivatization step prior to, and after, trypsin digestion. In order to control for possible variations between sample preparations, 15 N-labelled recombinant H2AX and H2A1B were spiked into the three samples. These samples were then derivatized and digested as normal and analysed in technical triplicate.…”

In vitro protein phosphorylation as a template for SRM method development

“…Данные методы широко применяются для анализа небольших молекул (метаболитов, интермедиатов химического синтеза), и в последнее время активно внедряются в протеомные исследования благодаря увеличению чувствительности и динамического диапазона по сравнению с обычным ВЭЖХ-МС/МС, в котором используется режим "полного сканирования" [14]. В основном SRM/MRM подходы реализуются на масс-спектрометрах с тройным квадруполем, но также была показана высокая эффективность применения LTQ-Orbitrap (pSRM) [15] и Q-Orbitrap (PRM) [16,17] …”