ABSTRACT
Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (â¼2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.
Subject(s)
Isoelectric Focusing/methods , Mass Spectrometry/methods , Peptides/analysis , Proteins/analysis , Proteomics/methods , Amino Acid Sequence , Animals , Cell Line, Tumor , Humans , Isoelectric Focusing/economics , Male , Mass Spectrometry/economics , Molecular Sequence Data , Prostatic Neoplasms/chemistry , Proteomics/economics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/analysisABSTRACT
The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.
Subject(s)
Biotin/isolation & purification , Chromatography, Affinity/methods , Receptors, Androgen/isolation & purification , Streptavidin/chemistry , Amino Acid Sequence , Biotin/metabolism , Biotinylation , Cell Line, Tumor , Humans , Mass Spectrometry/standards , Molecular Sequence Data , Peptide Fragments/chemistry , Protein Binding , Protein Interaction Mapping/methods , Proteomics , Receptors, Androgen/biosynthesis , Receptors, Androgen/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Reference Standards , Saccharomyces cerevisiaeABSTRACT
Mass spectrometry-based proteomics is typically performed using high performance liquid chromatography (HPLC) to introduce peptides into the instrument via electrospray ionization. A variety of configurations exist with varying degrees of precision and cost, but the ultimate goal is the reproducible delivery of peptides in well-separated elution peaks. It is well-known that the quality of chromatography can have a dramatic effect on sample identification as well as run-to-run reproducibility, which is especially important for quantitative analyses. Despite the importance of the HPLC system for both shotgun and targeted proteomics, there are few tools available to monitor HPLC performance. In this paper, we describe a new open-source software application, named ChromEval, to allow rapid assessment of HPLC performance, as well as to provide other metrics of mass spectrometer performance, including mass accuracy calibration. ChromEval permits the user to visually monitor the elution of a set of standard peptides in quality control runs interspersed among a regular workflow. To perform these tasks, ChromEval searches mzXML files using Tandem and presents the peptide results in a graphical user interface (GUI) that allows fast assessment of chromatography by visualization of superimposed elution peaks. This tool facilitates the identification and troubleshooting of chromatography problems such as retention time shifts and variance in sample loading due to autosampler error. It also provides crude but consistent metrics of instrument performance including mass accuracy calibration and number of peptides identified from the standard mixture. ChromEval generates easily interpretable data quickly and thereby enables go/no-go decision making during intensive instrument operation.
