Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTip(C18) microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTip(C18)-based purification of protein prior to MALDI-TOF-MS analysis.     

Optimization of separation and digestion conditions in immune complexome analysis

Immune complexome analysis is a method for identifying and profiling of antigens in circulating immune complexes (CICs); it involves separation of immune complexes from serum, direct tryptic digestion of these complexes, and protein analysis via nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS). To improve this method, we initially investigated the effects of two factors—the gradient elution program and nano-LC column type (C18-packed, C8-packed, or packed spray capillary column)—on the numbers of peptides and proteins identified. Longer gradient elution times resulted in higher identification capability throughout the range of 25–400 min. Moreover, the packed spray capillary column supported identification of more peptides and proteins than did any other column. In addition, microwave-assisted digestion was compared with conventional digestion, which involved incubation overnight at 37 °C. Microwave-assisted digestion produced more partially digested peptides than did conventional digestion. However, the percentages of miscleaved peptides in all of the identified peptides in microwave-assisted digestion of immune complexes (a protein mixture) were lower than those in the physical stimulation-assisted digestion of a model protein. Microwave-assisted digestion is slightly inferior to, or as effective as, conventional digestion, but it drastically reduces the digestion time.     

Determination of free and glucuronidated kaempferol in rat plasma by LC–MS/MS: Application to pharmacokinetic study

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid–liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm × 2.1 mm, 4.5 μm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min⁻¹. The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.     

Miniaturized solid-phase extraction and sample preparation for MALDI MS using a microfabricated integrated selective enrichment target

A microfabricated proteomic sample preparation and sample presentation device, Integrated Selective Enrichment Target, (ISET), comprising an array of 96 perforated nanovials is described. Each perforated nanovial can be filled with solid-phase extraction media for purification and concentration of peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The validity of the ISET sample preparation is shown by analysis of low nM-pM standard samples, as well as biological samples. The ISET solid-phase extraction sample preparation was compared to ZipTip and MassPREP PROtarget sample preparation, demonstrating a superior performance with respect to number of detected peptides and signal intensity of detected peptides.     

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles [1]. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species [2], [3], [4], [5], [6], [7], [8] and [9]. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans [2] and [3]. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.     

Comparison of methods to examine the endogenous peptides of fetal calf serum

There is a great desire to relate the patterns of endogenous peptides in blood to human disease and drug response. The best practices for the preparation of blood fluids for analysis are not clear and also relatively few of the peptides in blood have been identified by tandem mass spectrometry. We compared a number of sample preparation methods to extract endogenous peptides including C18 reversed phase, precipitation, and ultrafiltration. We examined the results of these sample preparation methods by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry (MS/MS) using MALDI-TOF/TOF and electrospray ionization-ion trap. Peptides from solid-phase extraction with C18 in the range of hundreds of femtomoles per spot were detected from the equivalent of 1 μL of serum by MALDI-TOF. We observed endogenous serum peptides from fibrinogen α- and β-chain, complement C3, α-2-HS-glycoprotein, albumin, serine (or cysteine) proteinase inhibitor, factor VIII, plasminogen, immunoglobulin, and other abundant blood proteins. However, we also recorded significant MS/MS spectra from tumor necrosis factor-α-, major histocompatibility complex-, and angiotensin-related peptides, as well as peptides from collagens and other low-abundance proteins. Amino acid substitutions were detected and a phosphorylated peptide was also observed. This is the first time the endogenous peptides of fetal serum have been examined by MS and where peptides from low-abundance proteins, phosphopeptides, and amino acid substitutions were detected.     

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

β-Hydroxymyristic acid as a chemical marker to detect endotoxins in dialysis water

An analytical chemical method has been developed for determination of β-hydroxymyristic acid (β-HMA), a component of lipopolysaccharides (LPSs/endotoxins) in dialysis water. In our investigation, the β-HMA component was used as a chemical marker for endotoxin presence in dialysis water because it is available in the molecular subunit (lipid A) and responsible for toxicity. It is the most abundant saturated fatty acid in that subunit. The developed method is based on fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD–PZ). A high-performance liquid chromatographic separation of the β-HMA derivative was achieved using an octadecyl silica column in gradient elution. A wide dynamic range of β-HMA was tested and a calibration curve was constructed with accuracy of 90% and variability of less than 10%. The limits of detection and quantification obtained were 2 and 5 μM, respectively. The developed method was applied to detect endotoxins in dialysis water by alkaline hydrolysis of LPS using NaOH (0.25 M) at 60 °C for 2 h. After hydrolysis, free acid was detected as its NBD–PZ derivative using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Good recovery rates ranging from 98 to 105% were obtained for β-HMA in dialysis water.     

Topology and dynamics of melittin within the liposome revealed by a combination of mass spectrometry and chemical modification

The topology and dynamics of melittin within the liposome were investigated by a mass spectrometry coupled with acetylation. The MALDI-TOF MS and MALDI-QIT-TOF MS/MS analyses revealed that only N-terminal amine of melittin was dominantly acetylated in the presence of liposome although all of four primary amines were completely and rapidly acetylated in aqueous solution. This result indicates that melittin adopts the N-terminal-outside transmembrane topology within the liposome. The time course of acetylation followed the first-order kinetics at any examined temperatures (6-30 °C). The rate constant was less than that of the acetylation of melittin in aqueous solution. The activation energy for acetylation (74 kJ mol⁻¹) was comparable to that for dissociation of a lipid monomer from the membrane, suggesting a float-like longitudinal motion of melittin within the liposome. These results demonstrate that a mass spectrometry combined with chemical modification is very efficient way for clarifying the topology and dynamics of peptides bound to the membrane.     

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Development of a high-throughput method for the determination of ethosuximide in human plasma by liquid chromatography mass spectrometry

A simple, rapid, sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of ethosuximide in human plasma is described. Analyte was chromatographed on a Hypersil Gold C₁₈ column (100 mm × 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow rate of 0.250 mL/min and pravastatin was used as the internal standard. The assay involves a simple solid-phase extraction procedure of 0.25 mL human plasma and the analysis was performed on a triple-quadrupole tandem mass spectrometer by MRM mode via electrospray ionization (ESI). The method was linear in the concentration range of 0.25–60.0 μg/mL. The lower limit of quantification (LLOQ) was 0.25 μg/mL. The within- and between-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 95.1% and 94.4% for ethosuximide and pravastatin, respectively. The analysis time for each sample was 1.8 min. The method was highly reproducible and gave peaks with excellent chromatography properties.     

Application of C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography–multiple reaction monitoring–tandem mass spectrometry (LC–MRM–MS/MS) method using ¹³C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting ¹³C-labeled peptides in blood sample with a C₁₈ cartridge, the extract was applied to a ¹³C monoisotopic transition LC–MRM–MS/MS system with d-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5–100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The ¹³C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three ¹³C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each ¹³C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC–MRM–MS/MS method.     

Enrichment of serum low-molecular-weight proteins using C18 absorbent under urea/dithiothreitol denatured environment

Serum low-molecular-weight proteins (LMWPs, molecular weight <30 kDa) are closely related to the body physiological and pathological situations, whereas many difficulties are encountered when enriching and fractionating them. Using C₁₈ absorbent (100 Å) enrichment and fractionation under urea/dithiothreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs could be enriched more than 100-fold and were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag (ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) levels, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and LTQ-Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). By virtue of its easy operation and high reproducibility to process large quantity complex serum samples, this method has potential uses in enriching LMWPs either in serum or in cell and tissue samples.     

Application of 13C stable isotope labeling liquid chromatography-multiple reaction monitoring-tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using (13)C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting (13)C-labeled peptides in blood sample with a C(18) cartridge, the extract was applied to a (13)C monoisotopic transition LC-MRM-MS/MS system with D-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5-100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The (13)C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three (13)C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each (13)C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method.     

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C₁₈ column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.     

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K⁺ as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.