Combining low and high mass ion accumulation for enhancing shotgun proteome analysis by accurate mass measurement

A simple procedure is described that increases sensitivity and dynamic range for the analysis of a proteome batch digest by FT-ICR mass spectrometry. Ions at the low and high mass ranges are preferentially collected using two different sets of tuning conditions. By combing data collected using tuning conditions that favor low mass (m/z < 2000) and high mass (m/z > 2000) ions, 277 proteins are identified for a whole cell lysate of Methanococcus maripaludis in a single HPLC-MALDI FT-ICR mass spectrometry experiment, a 70% improvement compared with previous analyses using a wide mass range acquisition. This procedure improves the detection of low abundance ions and thereby increases the range of proteins that are observed. Because the observed mass range is effectively narrower for each spectrum, mass calibration is more accurate than for the standard method that provides a wide range of masses. The trap plate potential on the analyzer cell may be set to a higher value than used for wide mass range measurements, increasing the ion capacity of the analyzer cell and extending the dynamic range, while still maintaining mass accuracy.     

Porous anodic alumina membrane as a sample support for MALDI-TOF MS analysis of salt-containing proteins

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analysis of proteins in salt-containing solution was performed for the first time using porous anodic alumina (PAA) membrane as sample support. The resulting spectral quality of proteins under standard sample preparation conditions was superior to that of normal metal sample stages. Analysis of phosphate-doped protein solutions indicated that porous anodic alumina membranes as a target yielded better results than a metallic target for salt-containing solutions. Because of the biocompatibility of the PAA, proteins can be adsorbed on the PAA and thus a washing process can be introduced to remove the salts from the PAA target before MS analysis. This desalting step significantly enhanced spectral quality, and better signal-to-noise ratios were obtained. The present technique is promising for proteomics research.     

Standard procedures for native CZE-MS of proteins and protein complexes up to 800 kDa

Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving “native” non-covalent inter- and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high-quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30–800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE-MS setup. This study is aimed to help normalize nCZE-MS practices to enhance the CE community and provide a resource for the production of reproducible and high-quality data.     

Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.     

肽质量指纹谱鉴定蛋白质时生物信息学分析条件的优化

为了优化肽质量指纹谱(peptide mass fingerprint,PMF)鉴定蛋白质的生物信息学分析条件。将牛碳酸酐酶2(carbonic anhydrase-2,CAH2)和人热休克蛋白70s(Hsp70s)进行2-DE分离、酶解,肽段经过MALDI-TOFMS分析得到PMF数据。选择Swissprot、MSDB、NCBInr、Random等数据库和MASCOT与MS-Fit搜索引擎,以牛CAH2为模型优化搜索参数,结果表明:Swissprot是适合做蛋白PMF分析的数据库;主要参数最佳设置为:漏切位点数为1个,肽质量容错数为±1Da,同时肽质量类型选择平均分子质量比单同位素质量更便于候选蛋白的筛选。最后用人Hsp70s蛋白的PMF数据检验优化条件,结果表明,所选择的数据库及参数是可靠的。     

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.     

MALDI imaging mass spectrometry of β‐ and γ‐crystallins in the ocular lens

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age‐related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age‐related modifications. Previous work demonstrated that, under standard protein IMS conditions, α‐crystallin signals dominated the mass spectrum and age‐related modifications to α‐crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β‐ and γ‐crystallins in ocular lens tissue. Acquired images showed that γ‐crystallins were localized predominately in the lens nucleus whereas β‐crystallins were primarily localized to the lens cortex. Age‐related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top‐down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC‐MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.     

Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry

Identifying unknown proteins has become a central focal point for proteomic and biopharmaceutical development laboratories. Our laboratory investigated using quadrupole time-of-flight mass spectrometry (Qq/TOFMS) for the analysis of intact proteins for the purpose of identifying unknowns while limiting the number of sample-handling steps between protein extraction and identification. Eight standard proteins, both unmodified and disulfide-bonded and ranging in mass from 5 to 66 kDa, were analyzed using nanoelectrospray and collision-induced dissociation to generate peptide sequence tags. An MS analysis, followed by MS/MS analyses on two to five individual protein charge states, were obtained to make an identification. Peptide sequence tags were extracted from the MS/MS data and used, in conjunction with molecular mass and source origin, to obtain protein identifications using the web-based search engine ProteinInfo (www.proteometrics.com). All of the proteins were unambiguously identified from the input data, after which, all of the major product ions were identified for structural information. In most cases, N- and/or C-terminal ions, and also stretches of consecutive product ions from the protein interior, were observed. This method was applied to the analysis and identification of an unknown detected via reversed-phase high-performance liquid chromatography.     

Application of fractional mass for the identification of peptide-oligonucleotide cross-links by mass spectrometry

A method has been developed to identify oligonucleotide-peptide heteroconjugates by accurate mass measurements using MS. The fractional mass (the decimal fraction mass value following the monoisotopic nominal mass) for peptides and oligonucleotides is different due to their differing molecular compositions. This property has been used to develop the general conditions necessary to differentiate peptides and oligonucleotides from oligonucleotide-peptide heteroconjugates. Peptides and oligonucleotides generated by the theoretical digestion of various proteins and nucleic acids were plotted as nominal mass versus fractional mass. Such plots reveal that three nucleotides cross-linked to a peptide produce enough change in the fractional mass to be recognized from non-cross-linked peptides at the same nominal mass. Experimentally, a Cytochrome c digest was spiked with an oligonucleotide-peptide heteroconjugate and conditions for analyzing the sample using liquid chromatography (LC)-MS were optimized. Upon analysis of this mixture, all detected masses were plotted on a fractional mass plot and the heteroconjugate could be readily distinguished from non-cross-linked peptides. The method developed here can be incorporated into a general proteomics-like scheme for identifying protein-nucleic acid cross-links, and this method is equally applicable to characterizing cross-links generated from protein-DNA and protein-RNA complexes.     

A novel Bicine running buffer system for doubled sodium dodecyl sulfate - polyacrylamide gel electrophoresis of membrane proteins

A novel, Bicine-based SDS-PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.     

Efficient enrichment and identification of phosphopeptides by cerium oxide using on-plate matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

An efficient and simple method for enrichment and identification of phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using cerium oxide is presented. After pretreatment of tryptic digests of phosphoproteins with CeO(2), nonphosphopeptides are discarded and phosphopeptides are enriched. By applying the separated CeO(2) on a target plate and analysis using MALDI-TOF MS, peaks of phosphopeptides and their correspondingly series of dephosphorylated peptides are observed in the mass spectra. Thus, the phosphopeptides are very easy to identify with the mass difference, which are all 80 Da between adjacent peaks in the same series, and clear background in the spectra owing to elimination of signal suppression from large amounts of nonphosphopeptides. Furthermore, the phosphopeptides can be dephosphorylated completely after a further NH(4)OH elution. Tryptic digest products from several standard proteins are pretreated using CeO(2) to demonstrate the efficiency of this method. Phosphopeptides from a very small quantity of human serum are enriched and analyzed, and proteins also identified by searching against a database using Mascot on MALDI-TOF/TOF fragments, which indicates that this method may be employed in complex samples for further application.     

Analysis of peptides using partial (no discharge) atmospheric pressure chemical ionization conditions with ion trap mass spectrometry

A novel approach, based on the use of atmospheric pressure chemical ionization ion trap mass spectrometry (APCI-ITMS) conditions, but without using corona discharge, was used to analyze peptides. The proposed method was applied to three standard peptides (bombesin, trityrosine and tyrosine-glycine-glycine) as well as peptides obtained through enzymatic digestion of two standard proteins (horse cytochrome c and horse myoglobin).     

固相微萃取-气相色谱/同位素质谱法测定水中挥发性有机物单体碳同位素

建立了采用75μm碳分子筛/聚二甲基硅氧烷(CAR-PDMS)纤维的固相微萃取-气相色谱/同位素质谱联用方法测定水中挥发性有机污染物碳同位素。使用浸入式固相微萃取和顶空固相微萃取方法进行实验确定在低浓度条件下最佳δ13C测试方法。通过使用顶空固相微萃取前处理技术进行单体同位素分析分析灵敏度更高,应用CSIA技术对1,2-二氯乙烯,三氯乙烯,四氯化碳进行单体同位素分析,方法的检出限为70μg/L,与样本的标准偏差小于0.3‰。该法适用于水体中微量挥发性有机污染物的同位素组成测定。     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.     

Selective isolation of N-terminal peptides from proteins and their de novo sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without regard to unblocking or blocking of N-terminal amino acids

We have developed a new method to determine the N-terminal amino acid sequences of proteins, regardless of whether their N-termini are modified. This method consists of the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling of sulfo-NHS-SS-biotin to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease followed by oxidation with performic acid; (4) specific isolation of N-terminal peptides from digests using DITC resins; (5) de novo sequence analysis of the N-terminal peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the CAF (chemically assisted fragmentation) method or tandem mass spectrometric (MS/MS) analysis according to unblocked or blocked peptides, respectively. By employing DITC resins instead of avidin resins used in our previous method (Yamaguchi et al., Rapid Commun. Mass Spectrom. 2007; 21: 3329), it has been possible to isolate selectively N-terminal peptides from proteins regardless of modification of N-terminal amino acids. Here we propose a universal method for N-terminal sequence analysis of proteins.     

高频感应燃烧红外线吸收法测定镍钛铌记忆合金中碳含量

建立高频感应燃烧–红外吸收测定镍钛铌记忆合金中碳含量的方法。对称样质量、助熔剂种类选择及加入量、空白坩埚的处理、仪器分析参数进行优化后,确定实验方案:称样质量0.5 g,助熔剂选用1.0 g钨锡粒,坩埚使用前于1 100℃中灼烧4 h后自然冷却,保存于干燥器中,仪器分析高频功率设定为1.54 k W。在选定的实验条件下,以钢铁标准样品绘制单点校准曲线,以钛合金标准物质IARM 271A验证曲线准确性,建立了高频燃烧红外吸收测定镍钛铌记忆合金中碳含量的方法。采用该方法分别对记忆合金样品NiTiNb–59炉、NiTiNb–40炉中碳含量进行测定,测定结果的相对标准偏差分别为3.42%,2.76%(n=10),在两样品中分别加入Leco501–501–1~#及AR871碳标准样品进行回收试验,回收率在96%~106%之间。该方法精密度好,准确性高,可用于镍钛铌记忆合金中碳含量的测定。     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.     

Liquid chromatography and electron-capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry

Liquid separation methods in combination with electrospray mass spectrometry as well as the recently introduced fragmentation method electron capture dissociation (ECD) have become powerful tools in proteomics research. This paper presents the results of the first successful attempts to combine liquid chromatography (LC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) with ECD in the analysis of a mixture of standard peptides and of a bovine serum albumin tryptic digest. A novel electron injection system provided conditions for ECD sufficient to yield extensive sequence information for the most abundant peptides in the mixtures on the time-scale of the chromatographic separation. The results suggest that LC/ECD-FTICRMS can be employed in the characterization of peptides in enzymatic digests of proteins or protein mixtures and identify and localize posttranslational modifications.     

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.