Development of MRM-based assays for the absolute quantitation of plasma proteins.

Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.

Developing an iMALDI method.

The iMALDI (immuno-MALDI) technique involves the affinity capture of target peptides from an enzymatic digest of a sample, followed by the direct analysis of the affinity beads while on a MALDI target. For determination of peptide concentration (and, by inference, protein concentration), stable-isotope-labeled standard peptides (SIS peptides) can be added to the digest and will be captured along with the native peptides. This technique can provide the highest possible specificity by determining two molecular characteristics of the epitope-containing peptides: (1) the molecular weight, typically measured to within 100 ppm or better by MALDI-MS, and (2) the amino acid sequence, by performing MALDI-MS/MS. This technique has been shown to be capable of detecting low-attomole levels of target peptides in environmental samples and in digests of human plasma. This chapter provides a detailed description of how to perform iMALDI analyses, starting with the selection of the target peptides. Examples are shown of the application of iMALDI to the detection of an organism that is a possible bioterrorism threat, and to the detection of two isoforms of human EGFR.

Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II.

CrkRS (Cdc2-related kinase, Arg/Ser), or cyclin-dependent kinase 12 (CKD12), is a serine/threonine kinase believed to coordinate transcription and RNA splicing. While CDK12/CrkRS complexes were known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II), the cyclin regulating this activity was not known. Using immunoprecipitation and mass spectrometry, we identified a 65-kDa isoform of cyclin K (cyclin K1) in endogenous CDK12/CrkRS protein complexes. We show that cyclin K1 complexes isolated from mammalian cells contain CDK12/CrkRS but do not contain CDK9, a presumed partner of cyclin K. Analysis of extensive RNA-Seq data shows that the 65-kDa cyclin K1 isoform is the predominantly expressed form across numerous tissue types. We also demonstrate that CDK12/CrkRS is dependent on cyclin K1 for its kinase activity and that small interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 has similar effects on the expression of a luciferase reporter gene. Our data suggest that cyclin K1 is the primary cyclin partner for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II.

The effect of pre-analytical variability on the measurement of MRM-MS-based mid- to high-abundance plasma protein biomarkers and a panel of cytokines.

Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.

The use of multiplexed MRM for the discovery of biomarkers to differentiate iron-deficiency anemia from anemia of inflammation.

In this study we demonstrate the use of a multiplexed MRM-based assay to distinguish among normal (NL) and iron-metabolism disorder mouse models, particularly, iron-deficiency anemia (IDA), inflammation (INFL), and inflammation and anemia (INFL+IDA). Our initial panel of potential biomarkers was based on the analysis of 14 proteins expressed by candidate genes involved in iron transport and metabolism. Based on this study, we were able to identify a panel of 8 biomarker proteins: apolipoprotein A4 (APO4), transferrin, transferrin receptor 1, ceruloplasmin, haptoglobin, lactoferrin, hemopexin, and matrix metalloproteinase-8 (MMP8) that clearly distinguish among the normal and disease models. Within this set of proteins, transferrin showed the best individual classification accuracy over all samples (72%) and within the NL group (94%). Compared to the best single-protein biomarker, transferrin, the use of the composite 8-protein biomarker panel improved the classification accuracy from 94% to 100% in the NL group, from 50% to 72% in the INFL group, from 66% to 96% in the IDA group, and from 79% to 83% in the INFL+IDA group. Based on these findings, validation of the utility of this potentially important biomarker panel in human samples in an effort to differentiate IDA, inflammation, and combinations thereof, is now warranted. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil.

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.

Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses.

Human pathogenic protozoa of the genus Leishmania undergo various developmental transitions during the infectious cycle that are triggered by changes in the host environment. How these parasites sense, transduce, and respond to these signals is only poorly understood. Here we used phosphoproteomic approaches to monitor signaling events in L. donovani axenic amastigotes, which may be important for intracellular parasite survival. LC-ESI-MS/MS analysis of IMAC-enriched phosphoprotein extracts identified 445 putative phosphoproteins in two independent biological experiments. Functional enrichment analysis allowed us to gain insight into parasite pathways that are regulated by protein phosphorylation and revealed significant enrichment in our data set of proteins whose biological functions are associated with protein turn-over, stress response, and signal transduction. LC-ESI-MS/MS analysis of TiO(2)-enriched phosphopeptides confirmed these results and identified 157 unique phosphopeptides covering 181 unique phosphorylation sites in 126 distinct proteins. Investigation of phosphorylation site conservation across related trypanosomatids and higher eukaryotes by multiple sequence alignment and cluster analysis revealed L. donovani-specific phosphoresidues in highly conserved proteins that share significant sequence homology to orthologs of the human host. These unique phosphorylation sites reveal important differences between host and parasite biology and post-translational protein regulation, which may be exploited for the design of novel anti-parasitic interventions.

A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin.

Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (∼80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.

Cytosolic protein interactions of the schizophrenia susceptibility gene dysbindin.

Using immunoprecipitation, mass spectrometry, and western blot analysis we investigated cytosolic protein interactions of the schizophrenia susceptibility gene dysbindin in mammalian cells. We identified novel interactions with members of the exocyst, dynactin and chaperonin containing T-complex protein complexes, and we confirmed interactions reported previously with all members of the biogenesis of lysosome-related organelles complex-1 and the adaptor-related protein complex 3. We report interactions between dysbindin and the exocyst and dynactin complex that confirm a link between two important schizophrenia susceptibility genes: dysbindin and disrupted-in-schizophrenia-1. To expand upon this network of interacting proteins we also investigated protein interactions for members of the exocyst and dynactin complexes in mammalian cells. Our results are consistent with the notion that impairment of aspects of the synaptic vesicle life cycle may be a pathogenic mechanism in schizophrenia.

Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies).

Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono- and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce.

A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers.

The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.

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 ([(13)C(6)]Arg or [(13)C(6)]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.

Identification of serum amyloid A as a biomarker to distinguish prostate cancer patients with bone lesions.

BACKGROUND: Prostate cancer has a propensity to metastasize to the bone. Currently, there are no curative treatments for this stage of the disease. Sensitive biomarkers that can be monitored in the blood to indicate the presence or development of bone metastases and/or response to therapies are lacking. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is an affinity-based approach that allows sensitive and high-throughput protein profiling and screening of biological samples. METHODS: We used SELDI-TOF MS for protein profiling of sera from prostate cancer patients (n = 38) with and without bone metastases in our effort to identify individual or multiple serum markers that may be of added benefit to those in current use. Serum was applied to ProteinChip surfaces (H4 and IMAC) to quickly screen samples and detect peaks predominating in the samples obtained from patients with bone metastases. Unique proteins in the bone metastasis cohort observed by SELDI-TOF MS were identified by two-dimensional gel electrophoresis, in-gel trypsin digestion, and tandem MS. The identities of the proteins were confirmed by ELISA and immunodepletion assays. RESULTS: The cluster of unique proteins in the sera of patients with bone metastases was identified as isoforms of serum amyloid A. Machine-learning algorithms were also used to identify patients with bone metastases with a sensitivity and specificity of 89.5%. CONCLUSIONS: SELDI-TOF MS protein profiling in combination with other proteomic approaches may provide diagnostic tools with potential clinical applications and serve as tools to aid in the discovery of biomarkers associated with various diseases.