ABSTRACT
The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.
ABSTRACT
In the past few years, mass spectrometry (MS) has emerged as an efficient tool for the multiplexed peptide and protein concentration determination by isotope dilution. Despite the growing use of isobaric tagging to perform relative quantitation for the discovery of potential biomarkers in biological fluids, no real application has so far been presented for their absolute quantitation. Isobaric tandem mass tags (TMTs) were used herein for the selection and quantitation of tryptic peptides derived from brain damage related proteins in cerebrospinal fluid (CSF). Proteotypic tryptic peptide analogues were synthesized, prepared in four reference amounts, differentially labeled with four isobaric TMTs with reporter-ions at m/z = 128.1, 129.1, 130.1, and 131.1, and mixed with CSF sample previously labeled with TMT 126.1. Off-gel electrophoresis (OGE) was used as first-dimension separation of the pooled sample. The resulting fractions were analyzed with reversed-phase liquid chromatography (RP-LC) tandem mass spectrometry (MS/MS), using tandem time-of-flight (TOF/TOF) and hybrid linear ion trap-orbitrap (LTQ-OT) instruments. Under collision-induced dissociation (CID) or higher-energy C-trap dissociation (HCD), the release of the reporter fragments from the TMT-labeled peptide standards provided an internal calibration curve to assess the concentration of these peptides in the CSF. This tool also allowed identifying selectively these peptides in CSF as only the targeted peptides showed specific fragmentation pattern in the TMT reporter-ion zone of the tandem mass spectra. Assays for the concentration measurements of peptides from PARK7, GSTP1, NDKA, and S100B proteins in CSF were further characterized using this novel, efficient, and straightforward approach.
Subject(s)
Chromatography, High Pressure Liquid/methods , Peptides/cerebrospinal fluid , Spectrometry, Mass, Electrospray Ionization/methods , Trypsin/metabolism , Amino Acid Sequence , Calibration , Chromatography, High Pressure Liquid/standards , Chromatography, Reverse-Phase , Glutathione S-Transferase pi/chemistry , Glutathione S-Transferase pi/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Molecular Sequence Data , Nerve Growth Factors/chemistry , Nerve Growth Factors/metabolism , Oncogene Proteins/chemistry , Oncogene Proteins/metabolism , Protein Deglycase DJ-1 , S100 Calcium Binding Protein beta Subunit , S100 Proteins/chemistry , S100 Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization/standardsABSTRACT
Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins. This procedure addresses a similar number of proteins, compared to the multidimensional protein identification technology approach, but offers additionally a quantitative analysis with its recently developed quantitative PST version.
Subject(s)
Proteome/analysis , Amino Acid Sequence , Animals , Carbon Isotopes , Fungal Proteins/analysis , Humans , Mass Spectrometry , Membrane Proteins/analysis , Mice , Molecular Sequence Data , Nuclear Proteins/analysis , Peptides/analysis , ProteomicsABSTRACT
A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.
