Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS

High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.     

Quantitative analysis of backbone-cyclised peptides in plants

Progress in understanding the biosynthetic pathway of the cyclotides has been hampered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r(2)>0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A(2)

Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.     

Isobaric Labeling and Data Normalization without Requiring Protein Quantitation

Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI,¹ the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 μg protein as the starting material.     

Determination of anti-HIV drug concentration in human plasma by MALDI-TOF/TOF

The antiretroviral therapeutic drug monitoring is becoming increased in clinical care to determine the best dosage regimen adapted to each patient. Here, the determination of the anti-HIV drugs lamivudine, lopinavir, and ritonavir concentration in the plasma of HIV-infected patients by MALDI-TOF/TOF is reported. The volume of the plasma sample was 600 microL. Plasma samples were extracted by solid-phase (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with methanol (100 microL), mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid), and spotted onto the MALDI-TOF/TOF sample target plate. The lamivudine, lopinavir and ritonavir concentration was determined by standard additions analysis. Regression of standard additions was linear over the anti-HIV drug concentration ranges explored (lamivudine, 0.010-1.0 pmol/microL; lopinavir and ritonavir, 0.0025-0.50 pmol/microL). Moreover, emtricitabine (i.e., the fluorinated analog of lamivudine) was used as the internal standard to determine the lamivudine concentration. The calibration curve was linear on the emtricitabine concentration ranging between 0.050 and 5.0 pmol/microL. The absolute recovery ranged between 80 and 110%. Values of the lamivudine, lopinavir and ritonavir concentration determined by MALDI-TOF/TOF are in excellent agreement with those obtained by HPLC-UV and HPLC-MS/MS. MALDI-TOF/TOF experiments allowed also the detection of the ritonavir metabolite R5. Zidovudine was undetectable by MALDI-TOF/TOF analysis because also the minimal laser intensity may induce the anti-HIV drug photolysis. The MALDI-TOF/TOF technique is useful to determine very low concentrations of anti-HIV drugs (0.0025-0.010 pmol/microL).     

Mixed-effects statistical model for comparative LC-MS proteomics studies

Comparing a protein's concentrations across two or more treatments is the focus of many proteomics studies. A frequent source of measurements for these comparisons is a mass spectrometry (MS) analysis of a protein's peptide ions separated by liquid chromatography (LC) following its enzymatic digestion. Alas, LC-MS identification and quantification of equimolar peptides can vary significantly due to their unequal digestion, separation, and ionization. This unequal measurability of peptides, the largest source of LC-MS nuisance variation, stymies confident comparison of a protein's concentration across treatments. Our objective is to introduce a mixed-effects statistical model for comparative LC-MS proteomics studies. We describe LC-MS peptide abundance with a linear model featuring pivotal terms that account for unequal peptide LC-MS measurability. We advance fitting this model to an often incomplete LC-MS data set with REstricted Maximum Likelihood (REML) estimation, producing estimates of model goodness-of-fit, treatment effects, standard errors, confidence intervals, and protein relative concentrations. We illustrate the model with an experiment featuring a known dilution series of a filamentous ascomycete fungus Trichoderma reesei protein mixture. For 781 of the 1546 T. reesei proteins with sufficient data coverage, the fitted mixed-effects models capably described the LC-MS measurements. The LC-MS measurability terms effectively accounted for this major source of uncertainty. Ninety percent of the relative concentration estimates were within 0.5-fold of the true relative concentrations. Akin to the common ratio method, this model also produced biased estimates, albeit less biased. Bias decreased significantly, both absolutely and relative to the ratio method, as the number of observed peptides per protein increased. Mixed-effects statistical modeling offers a flexible, well-established methodology for comparative proteomics studies integrating common experimental designs with LC-MS sample processing plans. It favorably accounts for the unequal LC-MS measurability of peptides and produces informative quantitative comparisons of a protein's concentration across treatments with objective measures of uncertainties.     

Internal calibrants allow high accuracy peptide matching between MALDI imaging MS and LC-MS/MS

One of the important challenges for MALDI imaging mass spectrometry (MALDI-IMS) is the unambiguous identification of measured analytes. One way to do this is to match tryptic peptide MALDI-IMS m/z values with LC-MS/MS identified m/z values. Matching using current MALDI-TOF/TOF MS instruments is difficult due to the variability of in situ time-of-flight (TOF) m/z measurements. This variability is currently addressed using external calibration, which limits achievable mass accuracy for MALDI-IMS and makes it difficult to match these data to downstream LC-MS/MS results. To overcome this challenge, the work presented here details a method for internally calibrating data sets generated from tryptic peptide MALDI-IMS on formalin-fixed paraffin-embedded sections of ovarian cancer. By calibrating all spectra to internal peak features the m/z error for matches made between MALDI-IMS m/z values and LC-MS/MS identified peptide m/z values was significantly reduced. This improvement was confirmed by follow up matching of LC-MS/MS spectra to in situ MS/MS spectra from the same m/z peak features. The sum of the data presented here indicates that internal calibrants should be a standard component of tryptic peptide MALDI-IMS experiments.     

A simple solid phase mass tagging approach for quantitative proteomics

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies.     

Quantitative approach to single-nucleotide polymorphism analysis using MALDI-TOF mass spectrometry

Pooling of DNA samples before genotyping is a valuable means of streamlining large-scale genotyping efforts in disease association studies, single-nucleotide polymorphism (SNP) validation or mutant allele screening programs. In this report, we explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantitative analysis of SNPs. The measurements are based on MALDI-TOF MS analysis of primer extension assays performed on standard mixtures of pooled PCR products at several test loci. The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation. The methods described maximize the sensitivity and quantitative capacity of MALDI-TOF MS while preserving the throughput and economic advantages of the MALDI-TOF platform. Using the format described, we demonstrate that allele frequencies as low as 5% can be detected quantitatively and unambiguously.     

Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100?000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14?000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.     

Quantitative analysis of free fatty acids in rat plasma using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with meso-tetrakis porphyrin as matrix

Quantitative analysis of free fatty acids was achieved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a meso-tetrakis porphyrin matrix. Cesium acetate was employed as a cationizing agent. The MALDI signal was reproducible and dominated by cesiated cesium carboxylates [RCOOCs + Cs]+. The addition of two Cs ions resulted in a mass shift of 264.8 Da for each fatty acid and greatly reduced background peaks. A linear relationship between fatty acid concentration and corresponding fatty acid to internal standard peak intensity ratio was observed for three representative fatty acids analyzed across a concentration range from 4.40 to 150 microM, with correlation coefficients between 0.986 and 0.987. The application of this method was demonstrated with the analysis of free fatty acids in nonfasted and fasted rat plasmas. A total of eight free fatty acids (14:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, and 20:4) were detected. The relative peak height ratios of the fatty acids to the internal standard allow quantitative measurements of the free fatty acids. It was shown that the levels of free fatty acids were higher in fasted rats than in rats in a nonfasted state. This method is simple, sensitive, and fast. Thus, it provides an appealing tool for the analysis of free fatty acids or other low-molecular weight compounds during drug discovery and/or development.     

Bayesian analysis of serial dilution assays

In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments.     

Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum: a novel approach for metabolic flux analysis.

In the present work, a novel comprehensive approach of (13)C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 microL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-(13)C glucose or with mixtures of (13)C6/(12)C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

Potential Pitfalls in MALDI-TOF MS Analysis of Abiotically Synthesized RNA Oligonucleotides

Demonstration of the abiotic polymerization of ribonucleotides under conditions consistent with conditions that may have existed on the prebiotic Earth is an important goal in “RNA world” research. Recent reports of abiotic RNA polymerization with and without catalysis rely on techniques such as HPLC, gel electrophoresis, and MALDI-TOF MS to analyze the reaction products. It is essential to understand the limitations of these techniques in order to accurately interpret the results of these analyses. In particular, techniques that rely on mass for peak identification may not be able to distinguish between a single, linear RNA oligomer and stable aggregates of smaller linear and/or cyclic RNA molecules. In the case of MALDI-TOF MS, additional complications may arise from formation of salt adducts and MALDI matrix complexes. This is especially true for abiotic RNA polymerization reactions because the concentration of longer RNA chains can be quite low and RNA, as a polyelectrolyte, is highly susceptible to adduct formation and aggregation. Here we focus on MALDI-TOF MS analysis of abiotic polymerization products of imidazole-activated AMP in the presence and absence of montmorillonite clay as a catalyst. A low molecular weight oligonucleotide standard designed for use in MALDI-TOF MS and a 3′-5′ polyadenosine monophosphate reference standard were also run for comparison and calibration. Clay-catalyzed reaction products of activated GMP and UMP were also examined. The results illustrate the ambiguities associated with assignment of m/z values in MALDI mass spectra and the need for accurate calibration of mass spectra and careful sample preparation to minimize the formation of adducts and other complications arising from the MALDI process.     

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.     

Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS

Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes.     

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC–MS/MS method in 17–19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC–MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.     

Calibration of bioaffinity assays using kinetic data

Bioaffinity assays are usually calibrated by using a set of standard measurements fitted to a simple empirical model. In this paper, a new calibration approach based on mechanistic model of reaction kinetics is presented. When the calibration assay is known in terms of reaction mechanism, incubation time, initial concentration, and rate constants, one can back-calculate concentrations of unknown samples measured in a nonequilibrium time point. This paper describes a calculation method of unknown sample concentrations based on kinetically measured single calibration assay point. The theoretical results are verified by two common in-vitro diagnostic assays.