Direct Analysis and Identification of Proteins in Mixtures by LC/MS/MS and Database Searching at the Low-Femtomole Level

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dualchannel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (Ͻ20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ionenergy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10 3 , and Ͼ10 5 for peptide abundances in the overall separation. (J Am Soc Mass Spectrom 2004, 15, 212-232)

Section: Direct Infusion Experimentsmentioning

confidence: 99%

“…While efforts to enhance the capabilities of 2D PAGE are underway, non-gel-based approaches for proteomic studies are being increasingly pursued [14]. Link et al [15] reported unbiased peptide-level identification of proteins from yeast (Saccharomyces cerevisiae), including many proteins with extremes in pI, MW, abundance, and hydrophobicity.…”

mentioning

confidence: 99%

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

Belov

Anderson

Wingerd

et al. 2004

“…Proteomic samples can be exceedingly complex-a proteolytic digest of a simple cell lysate can contain several hundred thousand peptides. Nanoscale capillary LC and electrospray ionization (ESI) coupled with data dependent MS/MS acquisition has been shown [1][2][3][4][5][6][7] to be an exceptionally useful tool for the analysis of complex proteomic samples, yielding high sensitivity (low femtomoles to high attomoles), high data acquisition rate (1000 precursors/h), and high information content (superior quality MS/MS spectra). Matrix assisted laser desorption ionization (MALDI) in conjunction with MS/MS analysis has also been used to acquire product ion spectra from proteomic samples, generating data sets both analogous to and complementary with those produced by LC/ESI/MS/MS [8 -12].…”

mentioning

confidence: 99%

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

Bodnar

Blackburn

Krise

et al. 2003

173

127

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/ MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques. (J Am Soc Mass Spectrom 2003, 14, 971-979)

“…Peptide mass fingerprinting determines the masses of all peptides present in the digestion mixture and compares the resulting patterns to those predicted for all entries in protein or genome sequence databases. This approach usually employs MALDI-MS because of its inherent simplicity, speed, and reliability, but is limited to digests of isolated proteins or simple protein mixtures [2].For more complex digestion mixtures, peptide sequencing using tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) is applied [3]. In this approach, individual peptides are separated, isolated from the mixture, and fragmented using low or high-energy collisions.…”

mentioning

confidence: 99%

“…For more complex digestion mixtures, peptide sequencing using tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) is applied [3]. In this approach, individual peptides are separated, isolated from the mixture, and fragmented using low or high-energy collisions.…”

mentioning

confidence: 99%

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation

Rogalski

Lin

Sniatynski

et al. 2005

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range. (MALDI) and electrospray ionization (ESI) based mass spectrometry has developed into an indispensable tool for researchers in molecular biology and biotechnology, and a key technology in proteomics [1]. Two different approaches incorporating mass spectrometry have emerged for the identification of proteins after enzymatic digestion into peptides. Peptide mass fingerprinting determines the masses of all peptides present in the digestion mixture and compares the resulting patterns to those predicted for all entries in protein or genome sequence databases. This approach usually employs MALDI-MS because of its inherent simplicity, speed, and reliability, but is limited to digests of isolated proteins or simple protein mixtures [2].For more complex digestion mixtures, peptide sequencing using tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) is applied [3]. In this approach, individual peptides are separated, isolated from the mixture, and fragmented using low or high-energy collisions. In order to identify the best-matching sequence, the fragmentation patterns obtained for a specific peptide are compared to those anticipated for each hypothetical peptide predicted from all database entries. Although recently introduced MALDI-MS/MS techniques now also permit selection and sequencing of individual peptides [4,5], the vast majority of peptide sequencing experiments are still carried out by ESI-MS/ MS. Particularly, the generation of intense fragment ions from multiply-charged peptide ions and the possibility of on-line separation b...