Making Broad Proteome Protein Measurements in 1−5 min Using High-Speed RPLC Separations and High-Accuracy Mass Measurements (original) (raw)
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Analytical Chemistry, 2005
The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through online electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-µm porous C18 particle-packed 50-µm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either ∼0.3 or ∼0.6 s for FTICR), peptide mass measurement accuracies of better than (15 ppm were obtained with the highspeed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than (15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13 000 dif-ferent putative peptides detected from a Shewanella oneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with ∼2000 different peptides from ∼600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.
Journal of the American Society for Mass Spectrometry, 2003
We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; ϳ3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, ϳ900 ORFs detected using LC-TOFMS, with ϳ500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of Ͻ3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized. (J Am Soc Mass Spectrom 2003, 14, 980 -991)
In silico analysis of accurate proteomics, complemented by selective isolation of peptides
Journal of Proteomics, 2011
Protein identification by mass spectrometry is mainly based on MS/MS spectra and the accuracy of molecular mass determination. However, the high complexity and dynamic ranges for any species of proteomic samples, surpass the separation capacity and detection power of the most advanced multidimensional liquid chromatographs and mass spectrometers. Only a tiny portion of signals is selected for MS/MS experiments and a still considerable number of them do not provide reliable peptide identification. In this article, an in silico analysis for a novel methodology of peptides and proteins identification is described. The approach is based on mass accuracy, isoelectric point (pI), retention time (t R ) and N-terminal amino acid determination as protein identification criteria regardless of high quality MS/MS spectra. When the methodology was combined with the selective isolation methods, the number of unique peptides and identified proteins increases. Finally, to demonstrate the feasibility of the methodology, an OFFGEL-LC-MS/MS experiment was also implemented. We compared the more reliable peptide identified with MS/MS information, and peptide identified with three experimental features (pI, t R , molecular mass). Also, two theoretical assumptions from MS/MS identification (selective isolation of peptides and N-terminal amino acid) were analyzed. Our results show that using the information provided by these features and selective isolation methods we could found the 93% of the high confidence protein identified by MS/MS with false-positive rate lower than 5%.
2003
We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, 900 ...
Journal of Chromatography A, 2009
This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30 mm × 0.32 mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.
Journal of Proteome Research, 2006
Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10 000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.
Angewandte Chemie, 2014
Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed-phase liquid chromatography (RPLC)-MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath-flow interface for the analysis of complex proteome digests. Single-shot CZE-MS/MS lead to the identification of over 10 000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100 min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state-ofthe-art nano ultrahigh-pressure LC system.
A simple approach for accurate peptide quantification in MS-based proteomics
Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spec-trometry (LC-MS/MS) and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and to avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along the sample preparation protocol, in particular after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in MS loading solvent, consuming only 2 microliter of sample. We determined the optimal peptide amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a d...