Quantification Of Protein Post-translational Modifications Research Articles

One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry.

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic “histone-like” peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

Rapid and reproducible phosphopeptide enrichment by tandem metal oxide affinity chromatography: application to boron deficiency induced phosphoproteomics.

Mass spectrometry has been instrumental in enabling the study of molecular signaling on a cellular scale by way of site-specific quantification of protein post-translational modifications, in particular phosphorylation. Here we describe an updated tandem metal oxide affinity chromatography (MOAC) combined phosphoprotein/phosphopeptide enrichment strategy, a scalable phosphoproteomics approach that allows rapid identification of thousands of phosphopeptides in plant materials. We implemented modifications to several steps of the original tandem MOAC procedure to increase the amount of quantified phosphopeptides and hence site-specific phosphorylation of proteins in a sample beginning with the less amounts of tissue and a substantially smaller amount of extracted protein. We applied this technology to generate time-resolved maps of boron signaling in Arabidopsis roots. We show that the successive enrichment of phosphoproteins in a first and phosphopeptide extraction in a second step using our optimized procedure strongly enriched the root phosphoproteome. Our results reveal that boron deficiency affects over 20% of the measured root phosphoproteome and that many phosphorylation sites with known biological function, and an even larger number of previously undescribed sites, are modified during the time course of boron deficiency. We identify transcription factors as key regulators of hormone signaling pathways that modulate gene expression in boron deprived plants. Furthermore, our phosphorylation kinetics data demonstrate that mitogen-activated protein kinase (MAPK) cascades mediate polarized transport of boron in Arabidopsis roots. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in plant biology research.

Inference and quantification of peptidoforms in large sample cohorts by SWATH-MS.

The consistent detection and quantification of protein post-translational modifications (PTMs) across sample cohorts is an essential prerequisite for the functional analysis of biological processes. Data-independent acquisition (DIA), a bottom-up mass spectrometry based proteomic strategy, exemplified by SWATH-MS, provides complete precursor and fragment ion information of a sample and thus, in principle, the information to identify peptidoforms, the modified variants of a peptide. However, due to the convoluted structure of DIA data sets the confident and systematic identification and quantification of peptidoforms has remained challenging. Here we present IPF (Inference of PeptidoForms), a fully automated algorithm that uses spectral libraries to query, validate and quantify peptidoforms in DIA data sets. The method was developed on data acquired by SWATH-MS and benchmarked using a synthetic phosphopeptide reference data set and phosphopeptide-enriched samples. The data indicate that IPF reduced false site-localization by more than 7-fold in comparison to previous approaches, while recovering 85.4% of the true signals. IPF was applied to detect and quantify peptidoforms carrying ten different types of PTMs in DIA data acquired from more than 200 samples of undepleted blood plasma of a human twin cohort. The data approportioned, for the first time, the contribution of heritable, environmental and longitudinal effects on the observed quantitative variability of specific modifications in blood plasma of a human population.

Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. II. Performance

In this report, we examine the performance of a mass spectrometry (MS)-based method for quantification of protein posttranslational modifications (PTMs) using stable isotope labeled internal standards. Uniform labeling of proteins and highly similar behavior of the labeled vs nonlabeled analyte pairs during chromatographic separation and electrospray ionization (ESI) provide the means to directly quantify a wide range of PTMs. In the companion report (Jiang et al., Anal. Biochem., 421 (2012) 506–516.), we provided principles and example applications of the method. Here we show satisfactory accuracy and precision for quantifying protein modifications by using the SILIS method when the analyses were performed on different types of mass spectrometers, such as ion-trap, time-of-flight (TOF), and quadrupole instruments. Additionally, the stable isotope labeled internal standard (SILIS) method demonstrated an extended linear range of quantification expressed in accurate quantification up to at least a 4 log concentration range on three different types of mass spectrometers. We also demonstrate that lengthy chromatographic separation is no longer required to obtain quality results, offering an opportunity to significantly shorten the method run time. The results indicate the potential of this methodology for rapid and large-scale assessment of multiple quality attributes of a therapeutic protein in a single analysis.

Quantification of protein posttranslational modifications using stable isotope and mass spectrometry: I: Principles and applications

With the increased attention to quality by design (QbD) for biopharmaceutical products, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass spectrometry (MS) based method to quantify a wide range of posttranslational modifications (PTMs) in recombinant proteins using stable isotope-labeled internal standard (SILIS). The SILIS was produced through metabolic labeling where 15N was uniformly introduced at every nitrogen atom in the studied proteins. To enhance the accuracy of the method, the levels of PTMs in SILIS were quantified using orthogonal analytical techniques. Digestion of an unknown sample mixed with SILIS generates a labeled and a nonlabeled version of each peptide. The nonlabeled and labeled counterparts coelute during RP-HPLC separation but exhibit a sufficient mass difference to be distinguished by MS detection. With the application of SILIS, numerous PTMs can be quantified in a single analysis based on the measured MS signal ratios of 15N-labeled versus the nonlabeled pairs. Several examples using microbial and mammalian-expressed recombinant proteins demonstrated the principle and utility of this method. The results indicate that SILIS is a valuable methodology in addressing CQAs for the QbD paradigm.