Selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads

A method for the selective enrichment of tryptophan-containing peptides from complex peptide mixtures such as protein digests is presented. It is based on the reversible reaction of tryptophan with malondialdehyde and trapping of the derivatized Trp-peptides on hydrazide beads via the free aldehyde group of the modified peptides. The peptides are subsequently recovered in their native form by specific cleavage reactions for further (mass spectrometric) analysis. The method was optimized and evaluated using a tryptic digest of a mixture of 10 model proteins, demonstrating a significant reduction in sample complexity while still allowing the identification of all proteins. The applicability of the tryptophan-specific enrichment procedure to complex biological samples is demonstrated for a total yeast cell lysate. Analysis of the processed fraction by 1D-LC-MS/MS confirms the specificity of the enrichment procedure, as more than 85% of the peptides recovered from the enrichment step contained tryptophan. The reduction in sample complexity also resulted in the identification of additional proteins in comparison to the untreated lysate.     

Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([¹³C₆]Arg or [¹³C₆]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.MS is capable of sensitive and accurate protein quantitation based on the quantitation of proteolytic peptides as surrogates for the corresponding intact proteins. Over the past 10 years, MS-based protein quantitation based on the analysis of peptides (in other words, based on “bottom-up” proteomics) has had a profound impact on how biological problems can be addressed (1, 2). Although advances in MS instrumentation have contributed to the improvement of MS-based protein quantitation, the use of stable isotopes in quantitative work flows has arguably had the greatest impact in improving the quality and reproducibility of MS-based protein quantitation (3–5).The ongoing development of untargeted MS-based quantitation work flows has focused on increasingly exhaustive sample prefractionation methods, at both the protein and peptide levels, with the goal of detecting and quantifying entire proteomes (6). Although untargeted MS-based quantitation work flows have their utility, they are costly in terms of lengthy MS data acquisition and analysis times, and as a result, they are often limited to quantifying differences between small sample sets (n < 10). To facilitate rapid quantitation of larger, clinically relevant sample sets (n > 100) there is a need to both simplify sample preparation and reduce MS analysis time.Multiple reaction monitoring (MRM)¹ is a tandem MS (MS/MS) scan mode unique to triple quadrupole MS instrumentation that is capable of rapid, sensitive, and specific quantitation of analytes in highly complex sample matrices (7). MRM is a targeted approach that requires knowledge of the molecular weight of an analyte and its fragmentation behavior under CID. MRM is capable of highly reproducible concentration determination when stable isotope-labeled internal standards are included in work flows and has been used for decades for the quantitation of low molecular mass analytes (<1000 Da) in pharmaceutical, clinical, and environmental applications (7, 8).The combination of triple quadrupole MS instrumentation with nanoliter flow rate high performance LC and nanoelectrospray ionization provides the necessary sensitivity for detection and quantitation of biological molecules such as peptides in complex samples such as plasma by MRM. When combined with the use of isotopically labeled synthetic peptide standards, MRM analysis is capable of sensitive (attomole level) and absolute determination of peptide concentrations across a wide concentration scale spanning a dynamic range of 10³–10⁴ (1, 9–13).Several recent studies involving MRM-based analysis of plasma proteins have focused on increasing MRM detection sensitivity by fractionating plasma using either multidimensional liquid chromatography, affinity depletion of high abundance proteins (11, 14, 15), or affinity enrichment of low abundance peptides (16, 17). Anderson and Hunter (14) have shown that LC-MRM/MS analysis is capable of detecting 47 moderate to high abundance proteins in plasma without depletion even though ∼90% of the total protein by weight in trypsin-digested plasma can be attributed to 10 high abundance proteins (18).Relative abundance of a protein does not preclude its involvement in disease. In fact, 32 of the 47 plasma proteins detected by Anderson and Hunter (14) have been implicated as putative markers for cardiovascular disease. The ability to rapidly quantify proteins in a highly multiplexed manner using MRM and internal standard peptides expands the potential application of MRM quantitation beyond biomarker validation and into the field of biomarker discovery. Targeted, simultaneous quantitation of hundreds of proteins in a single analysis will enable rapid protein expression profiling of large (n > 100) clinically relevant sample sets in a manner similar to DNA microarray expression profiling. By allowing researchers to look at patterns of expression levels of a large number of proteins in a large number of samples (as opposed to looking at the expression levels of only a single protein), multiplexed MRM-based quantitation will allow the correlation of expression patterns with particular diseases. Once these characteristic patterns have been established, physicians will be able to use these protein expression patterns to diagnose diseases in the same way they currently use blood chemistry panels or comprehensive metabolic panels.When considering the clinical utility of MS-based assays, direct comparisons are often made to ELISA, which is considered the “gold standard” for protein quantitation in clinical samples. Attributes of ELISAs, such as “time to first result” (1–2 h (19)) and the ability to quantify 96 or 384 samples in parallel because of their microtiter plate-based format, are currently difficult to match with MS-based protein assays. However, MRM protein assays may surpass ELISA in the rapid development of clinically useful, multiplexed protein assays. The impact of multiplexed assays in the field of genomics has increased interest in multiplexed quantitation of many proteins in individual clinical samples (19). Development and characterization of MRM-based protein assays using isotopically labeled peptides is rapid and inexpensive compared with the time and cost associated with the generation and characterization of antibodies for ELISA development.In this study, we describe the creation of a customizable mixture of concentration-balanced stable isotope-labeled standard (SIS) peptides representing an initial panel of 45 human plasma proteins. We used this mixture of SIS peptides to develop a suite of multiplexed, rapid, and reproducible MRM-based assays for expression profiling of these 45 proteins in simple tryptic digests of whole plasma. Additionally we characterized the analytical performance of these MRM peptide assays with respect to their reproducibility, and we demonstrated their utility for absolute protein concentration determination.Multiplexed MRM quantitation of peptides for protein quantitation has the potential to replace iTRAQ or other isotope label and label-free quantitative proteomics approaches because the approach is much faster than these other methods (30–60 min per analysis compared with 4 days for LC-MALDI-based iTRAQ), has greater reproducibility (CV <5% versus iTRAQ CV >20%), and enables absolute quantitation (concentration and copy number versus only x-fold up- or down-regulated). Additionally MRM-based quantitation with SIS peptides does not “miss” peptides because the SIS peptide must be detected in every sample: this means that if an endogenous peptide is not observed then it is below the limit of detection.     

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.     

Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.     

Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.     

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.     

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole‐sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple‐reaction monitoring (MRM) assays using nano‐liquid chromatography‐tandem mass spectrometry (nLC‐MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R² > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 ± 1.7 × 10³ proteins cell^?1 in the KB1^TM bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.     

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.     

Multiplexed MRM‐based quantitation of candidate cancer biomarker proteins in undepleted and non‐enriched human plasma

An emerging approach for multiplexed targeted proteomics involves bottom‐up LC‐MRM‐MS, with stable isotope‐labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer‐biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM‐based method. We have demonstrated excellent retention time reproducibility (median interday CV: 0.08%) and signal stability (median interday CV: 4.5% for the analytical platform and 6.1% for the bottom‐up workflow) for the 27 biomarker proteins (represented by 57 interference‐free peptides). The linear dynamic range for the MRM assays spanned four orders‐of‐magnitude, with 25 assays covering a 10³–10⁴ range in protein concentration. The lowest abundance quantifiable protein in our biomarker panel was insulin‐like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer‐associated protein biomarker panels in human plasma, prior to clinical use.     

MRM assay for quantitation of complement components in human blood plasma - a feasibility study on multiple sclerosis

As a proof-of-principle study, a multiple reaction monitoring (MRM) assay was developed for quantitation of proteotypic peptides, representing seven plasma proteins associated with inflammation (complement components and C-reactive protein). The assay development and the sample analysis were performed on a linear ion trap mass spectrometer. We were able to quantify 5 of the 7 target proteins in depleted plasma digests with reasonable reproducibility over a 2 orders of magnitude linear range (RSD≤25%). The assay panel was utilized for the analysis of a small multiple sclerosis sample cohort with 10 diseased and 8 control patients.     

Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.     

Accurate mass comparison coupled with two endopeptidases enables identification of protein termini

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.     

MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma

A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (<20% CV for 134/135 peptides) and no loss of retention time accuracy (<0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r >0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (<20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.     

Characterization of tetanus toxin, neat and in culture supernatant, by electrospray mass spectrometry

A method was developed for the liquid chromatographic-mass spectrometric (LC-MS) identification of extremely neurotoxic toxins. The method combines sample treatment in a safety containment and analysis of detoxified material in a common laboratory facility. The method was applied to the characterization of neat tetanus toxin and subsequent identification of the toxin in cell lysate supernatants and culture supernatants from different Clostridium tetani bacteria strains. Characterization of the neat toxin was accomplished by (1) accurate mass measurement of enzyme digest fragments of the toxin and (2) tandem mass spectrometric (MS/MS) amino acid sequencing of selected peptides. Accurate mass measurement proved no longer feasible for the analysis of supernatants, due to the overwhelming presence of peptides from proteins other than toxin. Even when high-molecular-weight proteins were filtered from the lysates and treated, the retained protein fraction yielded too many peptides. However, MS/MS could successfully be applied when the findings from the characterization of neat toxin were employed. Thus, LC-MS/MS of selected precursor ions from trypsin digest fragments yielded specific sequence data for identification of the toxin. This procedure provided reliable identification of the toxin at levels above 1 microg/ml and within a day. Investigations with the method developed will be extended to the botulinum neurotoxins.     

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.     

An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC: its application to protein peptide mapping by off-line HPLC-MALDI MS

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.     

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.     

Enhanced detection of low abundance human plasma proteins using a tandem IgY12-SuperMix immunoaffinity separation strategy

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates approximately 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one- and two-dimensional LC-MS/MS revealed a 60-80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of approximately 100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with >90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative.     

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.     

Simultaneous quantification of the new HIV protease inhibitors atazanavir and tipranavir in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl plasma for atazanavir and 50 microl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column (50 mm x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05-10 microg/ml for atazanavir and 0.1-75 microg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within +/-7.3 and +/-7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC-MS/MS assay for the quantification of protease inhibitors.