Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR)

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.     

Development of a high-throughput IMS-IMS-MS approach for analyzing mixtures of biomolecules

A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be +/-7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 h. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed.     

Advanced mass spectrometric methods for the rapid and quantitative characterization of proteomes

Progress is reviewed towards the development of a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of high performance separations and mass spectrometry. The approach uses high accuracy mass measurements from Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate peptide 'accurate mass tags' (AMTs) produced by global protein enzymatic digestions for a specific organism, tissue or cell type from 'potential mass tags' tentatively identified using conventional tandem mass spectrometry (MS/MS). This provides the basis for subsequent measurements without the need for MS/ MS. High resolution capillary liquid chromatography separations combined with high sensitivity, and high resolution accurate FTICR measurements are shown to be capable of characterizing peptide mixtures of more than 10(5) components. The strategy has been initially demonstrated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, high sensitivity, and the capability for stableisotope labeling methods for precise relative protein abundance measurements.Abbreviations: LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; AMT, accurate mass tag; PMT, potential mass tag; MMA, mass measurement accuracy; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ppm, parts per million.     

Rapid analysis of tryptically digested cerebrospinal fluid using capillary electrophoresis-electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry

Mass spectrometry has in recent years been established as the method of choice for protein identification and characterization in proteomics. Capillary electrophoresis (CE) is a fast and efficient method for the separation of peptides and proteins. The on-line combination of CE with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) has been shown to be a powerful tool in the analysis of complex mixtures of proteins. This paper presents the first results from a proteomic analysis of human cerebrospinal fluid proteins by tryptic digestion and CE-FTICR-MS, where 30 proteins could be identified on a 95% confidence level with mass measurement errors less than 5 ppm.     

Identification of phosphorylation sites of proteins by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry

The phosphorylation sites of two phosphorylated proteins, bovine β-casein and myelin basic protein (MBP), were identified by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry (HPLC-ESI-QITMS). The tryptic digest of each protein was separated by HPLC, the molecular weight of each peptide was determined by ESI-QITMS on line, and MS/MS spectrum of each peptide was simultaneously obtained by the combination of collision-induced desorption (CID) technique and tandem mass spectrometry (MS/MS) of QITMS. The phosphorylated peptide was identified by looking into whether the difference between the observed and predicted molecular weights of a peptide is 80 u or its integral multiple. Then the phosphorylation site was identified through manual interpretation of the MS/MS spectrum of the phosphorylated peptide or automatic SEQUEST data base-searching.     

Identification of phosphorylation sites of proteins by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry

The phosphorylation sites of two phosphorylated proteins, bovine β-casein and myelin basic protein (MBP), were identified by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry (HPLC-ESI-QITMS). The tryptic digest of each protein was separated by HPLC, the molecular weight of each peptide was determined by ESI-QITMS on line, and MS/MS spectrum of each peptide was simultaneously obtained by the combination of collision-induced desorption (CID) technique and tandem mass spectrometry (MS/MS) of QITMS. The phosphorylated peptide was identified by looking into whether the difference between the observed and predicted molecular weights of a peptide is 80 u or its integral multiple. Then the phosphorylation site was identified through manual interpretation of the MS/MS spectrum of the phosphorylated peptide or automatic SEQUEST data base-searching.     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.     

Plasma desorption mapping of the tryptic digest of 23-kDa recombinant human growth hormone

Plasma desorption mass spectrometry has been used to map the tryptic fragments from the 23-kDa recombinant human growth hormone protein. The unfractionated tryptic digest was adsorbed directly onto a nitrocellulose sample foil and mass spectra were obtained in both the positive and the negative ion mode. The adsorbed sample was then washed with deionized water and its mass spectrum was again obtained. The latter spectrum revealed tryptic fragments that were not observed in the spectra of the unwashed sample, which can be attributed (to some extent) to the removal of hydrophilic residues during washing. From this study a protocol, aimed at the complete mapping of tryptic fragments, is outlined.     

Proteomic analyses using an accurate mass and time tag strategy

An accurate mass and time (AMT) tag approach for proteomic analyses has been developed over the past several years to facilitate comprehensive high-throughput proteomic measurements. An AMT tag database for an organism, tissue, or cell line is established by initially performing standard shotgun proteomic analysis and, most importantly, by validating peptide identifications using the mass measurement accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) and liquid chromatography (LC) elution time constraint. Creation of an AMT tag database largely obviates the need for subsequent MS/MS analyses, and thus facilitates high-throughput analyses. The strength of this technology resides in the ability to achieve highly efficient and reproducible one-dimensional reversed-phased LC separations in conjunction with highly accurate mass measurements using FTICR MS. Recent improvements allow for the analysis of as little as picrogram amounts of proteome samples by minimizing sample handling and maximizing peptide recovery. The nanoproteomics platform has also demonstrated the ability to detect >10(6) differences in protein abundances and identify more abundant proteins from subpicogram amounts of samples. The AMT tag approach is poised to become a new standard technique for the in-depth and high-throughput analysis of complex organisms and clinical samples, with the potential to extend the analysis to a single mammalian cell.     

Identification of leptomeningeal metastasis-related proteins in cerebrospinal fluid of patients with breast cancer by a combination of MALDI-TOF, MALDI-FTICR and nanoLC-FTICR MS

Leptomeningeal metastasis (LM) is a devastating complication occurring in 5% of breast cancer patients. However, the current 'gold standard' of diagnosis, namely microscopic examination of the cerebrospinal fluid (CSF), is false-negative in 25% of patients at the first lumbar puncture. In a previous study, we analyzed a set of 151 CSF samples (tryptic digests) by MALDI-TOF and detected peptide masses that were differentially expressed in breast cancer patients with LM. In the present study, we obtain for a limited number of samples exact masses for these peptides by MALDI-FTICR MS measurements. Identification of these peptides was performed by electrospray FTICR MS after separation by nano-scale LC. The database results were confirmed by targeted high mass accuracy measurements of the fragment ions in the FTICR cell. The combination of automated high-throughput MALDI-TOF measurements and analysis by FTICR MS leads to the identification of 17 peptides corresponding to 9 proteins. These include proteins that are operative in host-disease interaction, inflammation and immune defense (serotransferrin, alpha 1-antichymotrypsin, hemopexin, haptoglobin and transthyretin). Several of these proteins have been mentioned in the literature in relation to cancer. The identified proteins alpha1-antichymotrypsin and apolipoprotein E have been described in relation to Alzheimer's disease and brain cancer.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.     

Infrared-assisted tryptic proteolysis for peptide mapping

In this report, infrared (IR) radiation was employed to enhance the efficiency of tryptic proteolysis for peptide mapping. Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of BSA and myoglobin (MYO) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 69% (BSA) and 90% (MYO) that were much better than those obtained by conventional in-solution tryptic digestion. The present IR-assisted proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.     

Liquid- and gas-phase nitration of bovine serum albumin studied by LC-MS and LC-MS/MS using monolithic columns

Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumin (BSA) and yielded sequence coverages of 95% and 33% with RP-HPLC--ESI--MS and RP-HPLC--ESI--MS/MS, respectively. At least 95% of the tyrosines were covered by the former method, whereas the latter method only detected less than 50% of the tyrosine-containing peptides. Upon liquid-phase nitration of BSA in aqueous solution using an excess of tetranitromethane, at least 16 of the 20 tyrosine residues were found to be nitrated. After exposure of solid BSA samples to gaseous nitrogen dioxide and ozone at atmospherically relevant concentration levels, only 3 nitrated peptides were detected. By use of such a model system, RP-HPLC--ESI--MS proved to be a rapid and highly efficient method for the comprehensive and quantitative detection of protein nitration.     

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.     

The use of accurate mass tags for high-throughput microbial proteomics

We describe and review progress towards a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements for microbial systems based upon the use of polypeptide accurate mass tags (AMTs) produced by global protein enzymatic digestions. The two-stage strategy exploits high accuracy mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, from potential mass tags tentatively identified using tandem mass spectrometry (MS/MS), providing the basis for subsequent measurements without the need for routine MS/MS. A high-resolution capillary liquid chromatography separation combined with high sensitivity, and high-resolution accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of more than 10(5) components, sufficient for broad protein identification using AMTs. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, and the capability for stable-isotope labeling methods for precise relative protein abundance measurements. The strategy has been initially evaluated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Additional developments, including the use of multiplexed-MS/MS capabilities and methods for dynamic range expansion of proteome measurements that promise to further extend the quality of proteomics measurements, are also described.     

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.