An improved clean sonoreactor-based method for protein identification by mass spectrometry-based techniques

A new clean fast (8 min) method for in-solution protein digestion without detergent or urea for protein identification by peptide mass fingerprint and mass spectrometry-based techniques is proposed. The new method avoids the use of time consuming desalting procedures entailing the following four steps done under the effect of an ultrasonic field provided by a sonoreactor: denaturation (1 min) in a mixed solution of water:acetonitrile 1/1 (v/v); protein reduction (1 min); protein alkylation (1 min); and protein digestion (5 min). Five proteins with masses comprised between 14.4 kDa and 97 kDa and the protein split-soret cytochrome c from D. desulfuricans ATCC27774, were successfully identified with this procedure. No differences were found in the sequence coverage or in the number of peptides matched when the new clean method was compared to another one using urea. Twofold better signal-to-noise ratios were obtained in the MALDI spectra from protein samples prepared with the new method when comparing it with a method using urea. The new digestion method avoids the need to remove salt content and increases throughput (six samples at once) while reducing sample loss and contamination from sample handling.     

Removal of sodium dodecyl sulfate from protein samples prior to matrix-assisted laser desorption/ionization mass spectrometry

Sodium dodecyl sulfate (SDS) is widely used for protein solubilization and for separation of proteins by SDS polyacrylamide gel electrophoresis (SDS-PAGE). However, SDS interferes with other techniques used for characterization of proteins, such as mass spectrometry (MS) and amino acid sequencing. In this paper, we have compared three procedures to remove SDS from proteins, including chloroform/methanol/water extraction (C/M/W), cold acetone extraction and desalting columns, in order to find a rapid and reproducible procedure that provides sufficient reduction of SDS and high recovery rates for proteins prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). A 1000-fold reduction of SDS concentration and a protein recovery at approximately 50% were obtained with the C/M/W procedure. The cold acetone procedure gave a 100-fold reduction of SDS and a protein recovery of approximately 80%. By using desalting columns, the removal of SDS was 100-fold, with a protein recovery of nearly 50%. Both the C/M/W and the cold acetone methods provided sufficient reduction of SDS, high recovery rates of protein and allowed the acquisition of MALDI spectra. The use of n-octyl-beta-D-glucopyranoside in the protein sample preparation enhanced the MALDI signal for protein samples containing more than 2 10(-4)% SDS, after the C/M/W extraction. Following the cold acetone procedure, the use of n-octylglucoside was found to be necessary in order to obtain spectra, but they were of lower quality than those obtained with the C/M/W method, probably due to higher residual amounts of SDS.     

Improved ultrasonic‐based sample treatment for the screening of anabolic steroids by gas chromatography/mass spectrometry

A rapid sample treatment procedure for the gas chromatography/mass spectrometry (GC/MS) determination of anabolic steroids in human urine has been developed. The new procedure makes use of ultrasonic energy to reduce reaction times and increase the overall sensitivity. The following variables affecting the performance of the ultrasonic treatment were optimised: (i) time, (ii) device, (iii) frequency, and (iv) temperature. It was found that, under an ultrasonic field, the hydrolysis of conjugated steroids with β‐glucuronidase from Escherichia coli K12 was possible with a treatment time of 10 min. The accuracy and precision of the ultrasonic method were found to be in agreement with those achieved with the conventional thermal conductivity procedure (Student's t‐test; p = 0.05, n = 10). After the enzymatic hydrolysis, the derivatisation of the target compounds with trimethylsilyl (TMS) reagent, methyl‐N‐trimethylsilyltrifluoroacetamide (MSTFA)/NH₄I/dithioerythritol (DTE) (1000:2:4, v/w/w), was also accelerated using ultrasonic energy. In order to test the applicability of the use of ultrasonic energy in the acceleration of the derivatisation reaction with TMS, the classic method of thermal conductivity was applied for comparative purposes to a pool of 35 androgenic anabolic steroids (AAS) and/or their metabolites. The results demonstrated that after 3 min of sonication in a Sonoreactor device (50% amplitude), 19 of the 35 compounds studied showed similar reaction yield to those obtained with the classic procedure requiring 30 min (Student's t‐test; p = 0.05, n = 5); 13 increased to higher silylation yields; and for the steroids 1‐testosterone, danazol and etiocholanolone‐D5, the same results were obtained using a sonication time of 5 min. The overall applicability of the ultrasonic‐based sample treatment method is shown by the analysis of five urine samples. The results are similar to those achieved by the routine procedure. The new method is fast, robust, and allows high sample throughput. Copyright © 2010 John Wiley & Sons, Ltd.     

New findings for in-gel digestion accelerated by high-intensity focused ultrasound for protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 microL. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 microg. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 microg/mL.     

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

Laser desorption/ionization mass spectrometry fingerprinting of complex hydrocarbon mixtures: application to crude oils using data mining techniques

Crude oil fingerprints were obtained from four crude oils by laser desorption/ionization mass spectrometry (LDI-MS) using a silver nitrate cationization reagent. Replicate analyses produced spectral data with a large number of features for each sample (>11,000 m/z values) which were statistically analyzed to extract useful information for their differentiation. Individual characteristic features from the data set were identified by a false discovery rate based feature selection procedure based on the analysis of variance models. The selected features were, in turn, evaluated using classification models. A substantially reduced set of 23 features was obtained through this procedure. One oil sample containing a high ratio of saturated/aromatic hydrocarbon content was easily distinguished from the others using this reduced set. The other three samples were more difficult to distinguish by LDI-MS using a silver cationization reagent; however, a minimal number of significant features were still identified for this purpose. Focus is placed on presenting this multivariate statistical method as a rapid and simple analytical procedure for classifying and distinguishing complex mixtures.     

Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry

A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic micro-reaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, non-specific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.     

Direct analysis of laser capture microdissected cells by MALDI mass spectrometry

Laser capture microdissection (LCM) has become an important tool in biological research, permitting isolation of specific cell populations from frozen tissue samples containing a mixture of cell types. Cells obtained by LCM can be directly analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). We report here methodology for the preparation and analysis of LCM captured cells with MALDI MS, giving high sensitivity and mass resolution. Comparison of the spectra obtained from cell populations of interest can identify unique disease or function-related protein markers. Using this approach, mass spectra obtained from human breast tissue containing invasive mammary carcinoma and normal breast epithelium using LCM were compared. Over 40 peaks were identified that significantly differed in intensity between invasive mammary carcinoma and normal breast epithelium. In addition, mass spectra are presented that show protein patterns from mouse liver and mouse colon crypts. The reported tissue preparation procedure and subsequent analysis by MALDI MS provide a new methodology for protein discovery involving LCM captured cells.     

Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS.     

Optimization of microfabricated nanoliter-scale solid-phase extraction device for detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization mass spectrometry

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins.     

Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based techniques

We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on ¹⁸O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant ¹⁸O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that ¹⁸O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.     

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry

Four methods were compared for analysis of host‐cell protein (HCP) impurities in a recombinant mAb. First, CZE‐MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE‐MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE‐ESI‐MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.     

Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry

Articular cartilage contains both chondrocyte cells and extracellular matrix (ECM) components. Currently, comprehensive information concerning the protein composition of human articular cartilage tissue is somewhat lacking. In this report we detail the use of tandem mass spectrometry (MS/MS) for a preliminary global identification of proteins from human articular knee cartilage tissue from patients diagnosed with osteoarthritis. Knee cartilage supernatant was fractionated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), in-gel digested and peptide sequences were then determined by performing on-line nano-liquid chromatography (LC)/MS/MS experiments using an ion trap mass spectrometer. Altogether, over 100 different proteins from nearly 700 unique peptide sequences were detected by MS/MS. The majority of the proteins identified are involved in ECM organization (35%), signal transduction and cell communication (14%), immune response (11%) and metabolism and energy pathways (11%). Proteins observed included several well-known cartilage components as well as lower abundant lesser known ECM proteins. Possible degradation products in the cartilage sample, such as from cartilage link protein, could also be detected by our mass spectrometry methods. We show here that mass spectrometry can be utilized as a tool for a fast, accurate and sensitive analysis of a complex mixture of cartilage proteins. It is believed that this type of proteomic analysis will aid future work centered on investigating the pathology of this and other related joint diseases.     

Sub part-per-million mass accuracy by using stepwise-external calibration in fourier transform ion cyclotron resonance mass spectrometry

A new external calibration procedure for FT-ICR mass spectrometry is presented, stepwise-external calibration. This method is demonstrated for MALDI analysis of peptide mixtures, but is applicable to any ionization method. For this procedure, the masses of analyte peaks are first accurately measured at a low trapping potential (0.63 V) using external calibration. These accurately determined (< 1 ppm accuracy) analyte peaks are used as internal calibrant points for a second mass spectrum that is acquired for the same sample at a higher trapping potential (1.0 V). The second mass spectrum has a approximately 10-fold improvement in detection dynamic range compared with the first spectrum acquired at a low trapping potential. A calibration equation that accounts for local and global space charge is shown to provide mass accuracy with external calibration that is nearly identical to that of internal calibration, without the drawbacks of experimental complexity or reduction of abundance dynamic range. For the 609 mass peaks measured using stepwise-external calibration method, the root-mean-square error is 0.9 ppm. The errors appear to have a Gaussian distribution; 99.3% of the mass errors are shown to lie within three times the sample standard deviation (2.6 ppm) of their true value.     

Restricted access chromatographic sample preparation of low mass proteins expressed in human fibroblast cells for proteomics analysis

Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the <20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOF-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein.     

Influence of one- and two-dimensional gel electrophoresis procedure on metal-protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.     

Ultrasonic-based protein quantitation by (18) O-labeling: optimization and comparison between different procedures

Herein we report results regarding the optimization and comparison between different ultrasonic‐based procedures for protein quantitation by the direct ¹⁸O‐labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme‐to‐protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 °C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.     

Qualitative drug analysis of hair extracts by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) is applied to a qualitative analysis of three sample extracts from hair suspected of containing various drug compounds. The samples were also subjected to a quantitative target analysis for codeine, morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methadone, and benzylpiperazine (BZP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). GC × GC/TOFMS provided a non-specific procedure that identified various drugs, metabolites, and impurities not included in the target analysis. They included cocaine, diazepam, and methaqualone (quaalude). Comprehensive GC × GC separation was achieved using twin-stage cryo-modulation to focus eluant from a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The TOF mass spectrometer provided unit mass resolution in the mass range m/z 5–1000 and rapid spectral acquisition (≤500 spectra/s). Clean mass spectra of the individual components were obtained using mass spectral deconvolution software. The ‘unknown’ components were identified by comparison with mass spectra stored in a library database.