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.     

Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.     

A robust biomarker discovery pipeline for high-performance mass spectrometry data

A high-throughput software pipeline for analyzing high-performance mass spectral data sets has been developed to facilitate rapid and accurate biomarker determination. The software exploits the mass precision and resolution of high-performance instrumentation, bypasses peak-finding steps, and instead uses discrete m/z data points to identify putative biomarkers. The technique is insensitive to peak shape, and works on overlapping and non-Gaussian peaks which can confound peak-finding algorithms. Methods are presented to assess data set quality and the suitability of groups of m/z values that map to peaks as potential biomarkers. The algorithm is demonstrated with serum mass spectra from patients with and without ovarian cancer. Biomarker candidates are identified and ranked by their ability to discriminate between cancer and noncancer conditions. Their discriminating power is tested by classifying unknowns using a simple distance calculation, and a sensitivity of 95.6% and a specificity of 97.1% are obtained. In contrast, the sensitivity of the ovarian cancer blood marker CA125 is approximately 50% for stage I/II and approximately 80% for stage III/IV cancers. While the generalizability of these markers is currently unknown, we have demonstrated the ability of our analytical package to extract biomarker candidates from high-performance mass spectral data.     

Physalaemin-like immunoreactive peptides from rabbit stomach

Two physalaemin (PHY)-like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP protonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment-matching program, indicated the amino acid sequences to be: (formula; see text) The sequences of PHLIPs-7 and -8 were confirmed with synthetic peptides. The PHY-antiserum cross-reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.     

Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography-tandem mass spectrometry

Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N(2)- and N(3)-deschloropropyl-dimethylifosfamide (N(2)-DCP-diMeIFO and N(3)-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289→168 for diMeIFO, m/z 213→168 for N(2)-DCP-diMeIFO, m/z 213→92 for N(3)-DCP-diMeIFO and m/z 261→154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20-10,000ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N(2)-DCP-diMeIFO, N(3)-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 μL plasma. The method was accurate with calculated bias from -5.8 to 4.0% for diMeIFO, from -1.1 to 10.6% for N(2)-DCP-diMeIFO and from -6.9 to 9.8% for N(3)-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.     

A data review and re-assessment of ovarian cancer serum proteomic profiling

Background

The early detection of ovarian cancer has the potential to dramatically reduce mortality. Recently, the use of mass spectrometry to develop profiles of patient serum proteins, combined with advanced data mining algorithms has been reported as a promising method to achieve this goal. In this report, we analyze the Ovarian Dataset 8-7-02 downloaded from the Clinical Proteomics Program Databank website, using nonparametric statistics and stepwise discriminant analysis to develop rules to diagnose patients, as well as to understand general patterns in the data that may guide future research.

Results

The mass spectrometry serum profiles derived from cancer and controls exhibited numerous statistical differences. For example, use of the Wilcoxon test in comparing the intensity at each of the 15,154 mass to charge (M/Z) values between the cancer and controls, resulted in the detection of 3,591 M/Z values whose intensities differed by a p-value of 10^-6 or less. The region containing the M/Z values of greatest statistical difference between cancer and controls occurred at M/Z values less than 500. For example the M/Z values of 2.7921478 and 245.53704 could be used to significantly separate the cancer from control groups. Three other sets of M/Z values were developed using a training set that could distinguish between cancer and control subjects in a test set with 100% sensitivity and specificity.

Conclusion

The ability to discriminate between cancer and control subjects based on the M/Z values of 2.7921478 and 245.53704 reveals the existence of a significant non-biologic experimental bias between these two groups. This bias may invalidate attempts to use this dataset to find patterns of reproducible diagnostic value. To minimize false discovery, results using mass spectrometry and data mining algorithms should be carefully reviewed and benchmarked with routine statistical methods.

     

In-source formation of N-acetyl-p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC-ECNICI-MS analysis

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M-PFB](-). In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen (N-acetyl-p-aminophenol, NAPAP-d(0); paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d(4); MW 155) in anhydrous acetonitrile and their GC-ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M-PFB](-) at m/z 150 from NAPAP-d(0) and m/z 154 from NAPAP-d(4), we observed highly reproducibly almost equally intense anions at m/z 149 and m/z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl-p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC-ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/z 149 to m/z 150 and of m/z 153 to m/z 154 decreased with increasing ion-source temperature in the range 100-250°C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI](-)) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC-EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC-MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.     

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).     

Covalent binding of 15-deoxy-delta12,14-prostaglandin J2 to PPARgamma

Since 15-deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2)) has been identified as an endogenous ligand of PPARgamma thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ(2) has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARgamma. We first observed that after incubation of 15dPGJ(2) with recombinant PPARgamma, the quantity of free 15dPGJ(2) measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARgamma(2)/15dPGJ(2) obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ(2). Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARgamma(2) ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z = 1314.699) corresponding to the addition of 15dPGJ(2) (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ(2) to PPARgamma, which opens up new perspectives to study the molecular basis for selective activities of PPARs.     

Determination of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostan oic acid and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoi c acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry

11 alpha-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M) and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m/z 349 and m/z 356 (2H7 standard) (daughter ions of m/z 637 and m/z 644 (2H7 standard] were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m/z 484 and m/z 491 (2H7 standard) with the parent ions m/z 682 and m/z 689 (2H7 standard) were chosen.     

Detection and characterization of N-alpha-chloramines by electrospray tandem mass spectrometry

Hypochlorous acid (HOCl) is a major product of activated neutrophils and may be important in antimicrobial activities of cells by oxidation or chlorination of susceptible amino acids. Three major peaks separated using C18 reverse phase-high-performance liquid chromatography RP-HPLC after incubation of leucine enkephalin (LeuEnk) with HOCl. Electrospray mass spectrometry showed masses of m/z 556.2, 590.2, and 624.4 corresponding to unmodified LeuEnk and peptides altered by addition of one or two chlorines (Cl). Formation of stable N-alpha-chloramines was indicated because the chlorinated peptides were readily reduced with the physiological reductants glutathione and ascorbic acid to LeuEnk (m/z 556.2) within 10 min. Sequence-specific ions observed in product ion spectra of single-charged monochlorinated and dichlorinated peptides were consistent with modification of the N-terminal amine. There was no evidence for chlorination of the Tyr aromatic ring in any spectra. Similar RP-HPLC profiles were obtained after oxidation of des-Tyr1-LeuEnk (GGFL) with the masses of the major products being m/z 393.3, 427.2, and 461.1. These were identified as unmodified GGFL, N-alpha-Cl-GGFL, and N-alpha-Cl2-GGFL based on comparison of tandem mass spectra. Oxidation of Met and formation of disulfide dimers was observed after incubation of either N-alpha-Cl-LeuEnk or N-alpha-Cl2-LeuEnk with a protein, indicating that both peptide N-alpha-chloramines were able to readily modify sulfur-containing amino acids within proteins. These data indicate initial formation of stable N-alpha-chorinated peptides after incubation with HOCl and suggest that N-alpha-chlorinated peptides may exist for some hours in the absence of physiological reducing agents or sulfur-containing amino acids.     

Split-field drift tube/mass spectrometry and isotopic labeling techniques for determination of single amino acid polymorphisms

A combination of split-field drift tube/mass spectrometry and isotopic labeling techniques is evaluated as a means of identifying single amino acid polymorphisms (SAAPs) in proteins. The method is demonstrated using cytochromec (equine and bovine) and hemoglobin (bovine and sheep). For these studies, proteins from different species are digested with trypsin, and the peptides are labeled at primary amine groups [using either a light (H(3))- or heavy (D(3))-isotopic reagent]. SAAP analysis is carried out by mixing the light-labeled peptides of one species with the heavy-labeled peptides of the other and electrospraying the resulting mixture into a split-field drift tube/mass spectrometer. Peptides having the same sequence in both species appear as doublets in the mass spectrum [shifted in mass-to-charge (m/z) according to the number of incorporated labels]; additionally, these species have identical mobility distributions. Peptides having sequences that differ by one amino acid appear as peaks in the mass spectrum that are shifted in m/z according to the mass difference associated with the SAAP and the number of incorporated labels. The ion mobility distributions for these peptides (differing by only a single amino acid) can often be rationalized by their expected similarities or differences providing additional evidence that they are related. In all, 12 and 26 peptide variants (between species) corresponding to 5 and 11 amino acid polymorphisms have been identified for the cytochrome c and hemoglobin protein samples, respectively.     

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

Background

The established methods of antenatal screening for Down syndrome are based on immunoassay for a panel of maternal serum biomarkers together with ultrasound measures. Recently, genetic analysis of maternal plasma cell free (cf) DNA has begun to be used but has a number of limitations including excessive turn-around time and cost. We aimed to develop an alternative method based on urinalysis that is simple, affordable and accurate.

Method

101 maternal urine samples sampled at 12–17 weeks gestation were taken from an archival collection of 2567 spot urines collected from women attending a prenatal screening clinic. 18 pregnancies in this set subsequently proved to be Down pregnancies. Samples were either neat urine or diluted between 10 to 1000 fold in dH₂O and subjected to matrix assisted laser desorption ionization (MALDI), time of flight (ToF) mass spectrometry (MS). Data profiles were examined in the region 6,000 to 14,000 m/z. Spectral data was normalised and quantitative characteristics of the profile were compared between Down and controls.

Results

In Down cases there were additional spectral profile peaks at 11,000-12,000 m/z and a corresponding reduction in intensity at 6,000-8,000 m/z. The ratio of the normalised values at these two ranges completely separated the 8 Down syndrome from the 39 controls at 12–14 weeks. Discrimination was poorer at 15–17 weeks where 3 of the 10 Down syndrome cases had values within the normal range.

Conclusions

Direct MALDI ToF mass spectral profiling of maternal urinary has the potential for an affordable, simple, accurate and rapid alternative to current Down syndrome screening protocols.     

Phospholipid analogues of Porphyromonas gingivalis

Porphyromonas has lipids containing hydroxy acids and C16:0 and iso-C15:0 major monocarboxylic acids among others. Nothing is known of its individual phospholipid molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species extracted from Porphyromonas gingivalis (seven strains), P. asaccharolytica (one strain) and P. endodontalis (two strains). Cultures on Blood-Fastidious Anaerobe Agar were harvested, washed and freeze-dried. Phospholipids were extracted and separated by fast atom bombardment mass spectrometry (FAB MS) in negative-ion mode. Phospholipid classes were also separated by thin layer chromatography (TLC). The major anions in the range m/z 209-299 were consistent with the presence of the C13: 0, C15: 0, C16: 0 and C18: 3 mono-carboxylate anions. Major polar lipid anion peaks in the range m/z 618-961 were consistent with the presence of molecular species of phosphatidylethanolamine, phosphatidylglycerol and with unidentified lipid analogues. Porphyromonas gingivalis differed from comparison strains of other species by having major anions with m/z 932, 946 and 960. Unusually, a feline strain of P. gingivalis had a major peak of m/z 736. Selected anions were studied by tandem FAB MS which revealed that peaks with m/z 653 and 946 did not correspond to commonly occurring classes of polar lipids. They were however, glycerophosphates. It is concluded that the polar lipid analogue profiles obtained with Porphyromonas are quite different from those of the genera Prevotella and Bacteroides but reveal heterogeneity within P. gingivalis.     

Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein.

Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carbon monoxide and 2-protocatechuoyl-phloroglucinol carboxylic acid, was purified from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glycan chains. The enzyme was resolved in SDS polyacrylamide gels in a diffuse protein band that corresponded to a molecular mass of 130-170 kDa. When purified flavonol 2,4-dioxygenase was heated, it dissociated into three peptides with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-35 kDa (S), which occurred in a molar ratio of 1:1:1, suggesting a LMS structure. Crosslinking led to a 90-97 kDa species, concomitant with the decrease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) peptides. Analysis by matrix-assisted laser desorption/ionization-time of flight-MS showed peaks at m/z approximately 69 600, m/z approximately 51 700, and m/z approximately 26 500 which are presumed to represent the three peptides of flavonol 2,4-dioxygenase, and a broad peak at m/z approximately 96 300, which might correspond to the LS heterodimer as formed in the crosslinking reaction. Based on the estimated molecular mass of 148 kDa, 1 mol of enzyme contained 1.0-1.6 mol of copper. Ethylxanthate, which specifically reduces CuII to CuI ethylxanthate, is a potent inhibitor of flavonol 2,4-dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphenylthiocarbazone) strongly inhibited the enzymatic activity, but inactivation was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-dioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 CuII protein. The Cu2+ is assumed to interact with four nitrogen ligands, and the CuII complex has a (distorted) square planar geometry.     

Microbore HPLC/mass spectrometry for the analysis of peptide mixtures using a continuous flow interface

Microbore HPLC techniques have been combined with fast atom bombardment mass spectrometry to provide HPLC/MS capabilities for the analysis of mixtures of peptides and small proteins. The interface between the liquid chromatograph and mass spectrometer is a continuous flow direct insertion probe which contains a fused silica capillary that delivers the eluting solvent to the FAB source of the mass spectrometer at a rate of 5-10 microL/min. Data are presented for the analysis of several mixtures of peptides ranging in molecular weights from about 900 to 6000 daltons. In addition, the analysis of 100 pmol of a tryptic digest of whale myoglobin is shown where 16 of the possible 19 peptides were identified in the mass range m/z 2200-250. The advantages of this approach to HPLC/MS are a relatively high sensitivity because of the low flow rates and low background, and the ability to detect high molecular weight compounds.     

Investigations of animal blood samples after fragrance drug inhalation by gas chromatography/mass spectrometry with chemical ionization and selected ion monitoring.

The fragrance compounds linalool (1) and linalyl acetate (2) could be detected, identified and quantified (1: 7-9 ng ml-1; and 2: 1-2 ng ml-1 and 4-5 ng ml-1 as free linalool) in blood samples after inhalation in animal experiments (mice) by gas chromatography/mass spectrometry (GC/MS) with chemical ionization (CI) (ammonia); selected ion monitoring (SIM) mode (1: m/z 81, 137 and 154; 2: 47, 57 and 137) and GC/flame ionization detection (FID). The inhalation of these monoterpenes in concentrations of 5 mg l-1 air leads to a significant reduction of the motility of the test animals down to 30-40% with respect to the control group.     

Peptidomics of the pars intercerebralis-corpus cardiacum complex of the migratory locust, Locusta migratoria.

The pars intercerebralis-corpora cardiaca system (PI-CC) of insects is the endocrinological equivalent of the hypothalamus-pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI-TOF and electrospray ionization double quadrupole time of flight (ESI-Qq-TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI-TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.     

Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.     

Quantification of acetaminophen (paracetamol) in human plasma and urine by stable isotope-dilution GC-MS and GC-MS/MS as pentafluorobenzyl ether derivative

We report on the quantitative determination of acetaminophen (paracetamol; NAPAP-d(0)) in human plasma and urine by GC-MS and GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode after derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br). Commercially available tetradeuterated acetaminophen (NAPAP-d(4)) was used as the internal standard. NAPAP-d(0) and NAPAP-d(4) were extracted from 100-μL aliquots of plasma and urine with 300 μL ethyl acetate (EA) by vortexing (60s). After centrifugation the EA phase was collected, the solvent was removed under a stream of nitrogen gas, and the residue was reconstituted in acetonitrile (MeCN, 100 μL). PFB-Br (10 μL, 30 vol% in MeCN) and N,N-diisopropylethylamine (10 μL) were added and the mixture was incubated for 60 min at 30 °C. Then, solvents and reagents were removed under nitrogen and the residue was taken up with 1000 μL of toluene, from which 1-μL aliquots were injected in the splitless mode. GC-MS quantification was performed by selected-ion monitoring ions due to [M-PFB](-) and [M-PFB-H](-), m/z 150 and m/z 149 for NAPAP-d(0) and m/z 154 and m/z 153 for NAPAP-d(4), respectively. GC-MS/MS quantification was performed by selected-reaction monitoring the transition m/z 150 → m/z 107 and m/z 149 → m/z 134 for NAPAP-d(0) and m/z 154 → m/z 111 and m/z 153 → m/z 138 for NAPAP-d(4). The method was validated for human plasma (range, 0-130 μM NAPAP-d(0)) and urine (range, 0-1300 μM NAPAP-d(0)). Accuracy (recovery, %) ranged between 89 and 119%, and imprecision (RSD, %) was below 19% in these matrices and ranges. A close correlation (r>0.999) was found between the concentrations measured by GC-MS and GC-MS/MS. By this method, acetaminophen can be reliably quantified in small plasma and urine sample volumes (e.g., 10 μL). The analytical performance of the method makes it especially useful in pediatrics.