Protein sequencing by tandem mass spectrometry.

Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.     

Vitamin D and metabolites measurement by tandem mass spectrometry

The prevalence of vitamin D deficiency in the general population has become a major public health problem. Vitamin D deficiency might have significant consequences not only to bone health but possibly to autoimmune-, infectious and cardiovascular disease. This has resulted in increased clinical testing for 25-hydroxyvitamin D (25(OH)D) in serum, as circulating 25(OH)D is regarded as the best indicator of adequate exposure to sunlight and dietary intake of vitamin D. There are reportedly over 50 vitamin D metabolites of which 25(OH)D and 1,25(OH)2D are well known to provide clinical information. More recently, there is increasing interest in measuring the C3-epimer of 25(OH)D, which has shown to contribute significantly to the 25(OH)D concentration, particularly in infant populations, and in 24,25(OH)2D, a major catabolite of 25(OH)D metabolism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an analytical tool that allows the specific determination of all relevant vitamin D metabolites, with the potential of performing multiple analyte analysis in a single experimental setting, creating a vitamin D profile. This article reviews recent advances in the quantification of vitamin D metabolites using LC-MS/MS.     

串联质谱技术对苯丙酮尿症杂合子的检测

目的建立串联质谱技术测定血苯丙氨酸(Phe)和酪氨酸(Tyr)浓度方法,观察苯丙酮尿症(PKU)、PKU杂合子和正常对照组血Phe、Tyr浓度及其比值(Phe/Tyr)变化。方法研究对象为52例PKU患儿,152例PKU杂合子成人,160名正常成人作为对照。研究方法采用干血滤纸片法,血滤纸片经含已知浓度Phe和Tyr内标的甲醇萃取,盐酸正丁醇衍生后,用串联质谱仪分析。结果(1)样品回收率Phe在346μmol/L和485μmol/L浓度时分别为102%和100%,Tyr在276μmol/L和442μmol/L浓度时分别为103%和98%。(2)批内CVPhe为6.7%,Tyr为10.5%,批间CVPhe为12.2%,Tyr为9.9%。(3)PKU患者血Phe、Tyr浓度和Phe/Tyr值分别为(634.0±300.3)μmol/L、(41.9±16.3)μmol/L和16.5±9.9,PKU杂合子分别为(68.3±21.4)μmol/L、(40.1±11.8)μmol/L和1.7±0.4,对照组分别为(53.2±10.2)μmol/L、(43.6±9.5)μmol/L和1.2±0.2。PKU患者血Phe、Phe/Tyr值与PKU杂合子、对照组比较差异有统计学意义(P<0.01)。PKU杂合子与对照组比较,Phe、Phe/Tyr水平较高(均P<0.01),Tyr水平较低(P<0.01)。结论串联质谱技术能够准确测定干血滤纸片中Phe和Tyr浓度。PKU杂合子Phe浓度和Phe/Tyr值高于正常成人,但与正常人有明显的重叠。     

Peptide mixture sequencing by tandem Fourier-transform mass spectrometry.

Picomole samples of the linear peptide gramicidin D and cyclic peptide gramicidin S are shown to be impure by the laser-desorption formation of multiple groups of molecular adduct peaks by using Fourier-transform mass spectrometry. Selective excitation of the molecular peaks of the major sample component followed by collisionally activated dissociation provides complete sequence information for the cyclic decapeptide and for 12 of the 15 amino acids of the linear peptide. This instrumentation shows striking advantages in sensitivity, resolution, and mass accuracy in comparison to tandem mass spectrometers used previously.     

Detection of neonatal argininosuccinate lyase deficiency by serum tandem mass spectrometry

Argininosuccinate lyase (ASL) deficiency (McKusick 207900) is an inborn error of the urea cycle. The leading symptom is progressive hyperammonaemia, which is a life-threatening condition, particularly in patients with a neonatal onset. Early diagnosis and treatment of the hyperammonaemia are necessary to improve survival and the long-term outcome of ASL-deficient patients. Currently, the diagnosis of ASL deficiency is based on the measurement of urea cycle intermediates and amino acids by automated quantitative ion exchange chromatography in plasma and urine. Here, we report a newborn presenting with coma and severe hyperammonaemia. ASL deficiency was suspected on the basis of an adapted tandem mass spectrometric (MS-MS) procedure which allows determination of argininosuccinate in addition to the amino acids in serum samples. MS-MS measurements revealed a characteristic increase of argininosuccinate, a moderate increase of citrulline, and lowered levels of arginine and ornithine in the serum of the patient. The diagnosis was confirmed by the detection of a novel homozygous frameshift mutation in exon 14 of the argininosuccinate lyase gene. We propose MS-MS as a diagnostic tool suitable for the rapid detection of specific alterations in the amino acid spectra caused by ASL deficiency.     

Screening blood spots for argininosuccinase deficiency by electrospray tandem mass spectrometry

Argininosuccinase deficiency is relatively more common in Saudi Arabia than other urea cycle detects (UCD) and its presentation is usually acute and virtually identical to the clinical presentation of other UCD. We developed a rapid, sensitive, and specific screening method for the diagnosis of argininosuccinase deficiency from blood spots. using electrospray tandem mass spectrometry. A 96-well microplate batch process is used for extraction of argininosuccinic acid (ASA), other amino acids and acylcarnitines (Rashed et al. 1995). ASA and other metabolites are derivatized to the corresponding butyl derivatives. The tris-butyl ester of ASA (MH = 459.3) yields two major fragments at m/z 70 and m/z 144 under mild collision induced collision. montitored in the product ion spectrum using a narrow mass range (65-150 kDa). A processing algorithm "CAMPA" is used to automatically calculate the height ratios of selected masses and flags data files as "abnormal" when certain threshold is exceeded. The method is integrated with our existing 2-minute MS/MS method for profiling amino acids and acylcarnitines (Rashed et al. 1997). Using this approach for two years we diagnosed 16 ALD cases from 14 hyperammonemic infants, one high-risk newborn, and one from a regular newborn screening blood spot.     

Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry

A de novo sequencing program for proteins is described that uses tandem MS data from electron capture dissociation and collisionally activated dissociation of electrosprayed protein ions. Computer automation is used to convert the fragment ion mass values derived from these spectra into the most probable protein sequence, without distinguishing Leu/Ile. Minimum human input is necessary for the data reduction and interpretation. No extra chemistry is necessary to distinguish N- and C-terminal fragments in the mass spectra, as this is determined from the electron capture dissociation data. With parts-per-million mass accuracy (now available by using higher field Fourier transform MS instruments), the complete sequences of ubiquitin (8.6 kDa) and melittin (2.8 kDa) were predicted correctly by the program. The data available also provided 91% of the cytochrome c (12.4 kDa) sequence (essentially complete except for the tandem MS-resistant region K(13)-V(20) that contains the cyclic heme). Uncorrected mass values from a 6-T instrument still gave 86% of the sequence for ubiquitin, except for distinguishing Gln/Lys. Extensive sequencing of larger proteins should be possible by applying the algorithm to pieces of approximately 10-kDa size, such as products of limited proteolysis.     

Identification of membrane proteins by tandem mass spectrometry of protein ions

The most common way of identifying proteins in proteomic analyses is to use short segments of sequence ("tags") determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning alpha-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1-4 transmembrane alpha-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5-18 transmembrane alpha-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase.     

High-resolution tandem mass spectrometry of large biomolecules.

Unit-resolution mass spectra have been obtained for peptides as large as 17 kDa, providing information on impurities and adduct ions, as well as accurate molecular weight values. Electrospray ionization produces many multiply-charged species of the same mass; isotopic peak resolution provides direct charge state assignment from the unit mass spacing of the isotopes. This is of special value when the spectrum also has many masses, such as from precursor ion dissociation or impurities. Mass measuring errors not only are concomitantly lower (less than 0.1 Da) than when the isotopic peaks are unresolved but also are independent of variations in 13C/12C natural isotopic abundances. Also, larger errors are avoided that occur when the measured peak envelope includes impurity or adduct ions. This also benefits tandem mass spectrometry; dissociation of peptide ions as large as 8.5 kDa yields fragment masses consistent (less than 0.1 Da) with their amino acid sequences.     

Nationwide survey of extended newborn screening by tandem mass spectrometry in Taiwan

In Taiwan, during the period March 2000 to June 2009, 1,495,132 neonates were screened for phenylketonuria (PKU) and homocystinuria (HCU), and 1,321,123 neonates were screened for maple syrup urine disease (MSUD), methylmalonic academia (MMA), medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, isovaleric academia (IVA), and glutaric aciduria type 1 (GA-1) using tandem mass spectrometry (MS/MS). In a pilot study, 592,717 neonates were screened for citrullinemia, 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC) and other fatty acid oxidation defects in the MS/MS newborn screening. A total of 170 newborns and four mothers were confirmed to have inborn errors of metabolism. The overall incidence was approximately 1/5,882 (1/6,219 without mothers). The most common inborn errors were defects of phenylalanine metabolism [five classic PKU, 20 mild PKU, 40 mild hyperphenylalaninemia (HPA), and 13 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency]. MSUD was the second most common amino acidopathy and, significantly, most MSUD patients (10/13) belonged to the Austronesian aboriginal tribes of southern Taiwan. The most frequently detected among organic acid disorders was 3-MCC deficiency (14 newborns and four mothers). GA-1 and MMA were the second most common organic acid disorders (13 and 13 newborns, respectively). In fatty acid disorders, five carnitine transport defect (CTD), five short-chain acyl-CoA dehydrogenase deficiency (SCAD), and two medium-chain acyl-CoA dehydrogenase (MCAD) deficiency were confirmed. This is the largest case of MS/MS newborn screening in an East-Asian population to date. We hereby report the incidences and outcomes of metabolic inborn error diseases found in our nationwide MS/MS newborn screening program.     

基于多维液相色谱质谱组合分析的痢疾杆菌蛋白质基因组学

目的 应用多维液相色谱质谱组合体系为基础的蛋白质组学方法对福氏痢疾杆菌基因组注释进行完善。方法 痢疾杆菌福氏2a型301株(Sf2a301)的全菌蛋白经胰酶消化,二维液相色谱分离后进行MALDI-TOF/TOF和ESI-MS/MS组合鉴定,质谱数据分别应用MASCOT和SEQUEST软件检索基于Sf2a301全基因组构建的6个读码框数据库,完成对原基因组注释的验证和补充。结果 研究表明多维液相色谱质谱组合体系能够增加鉴定蛋白的覆盖率,共鉴定Sf2a301的1 231个蛋白编码基因产物,涵盖了COGs 数据库22个功能分类组中的20个,包含306个功能未知的假定蛋白。发现了9个未注释的基因,得到RT-PCR和Northern blot的进一步验证。新基因大多数是重叠基因,包含3个嵌套基因。结论 多维液相色谱质谱组合体系相对于单一的串联质谱技术能够更加有效验证、补充痢疾杆菌的基因组注释,更新后的基因组注释库为今后开展痢疾杆菌功能研究提供更多的靶点。     

Expanded newborn screening by tandem mass spectrometry: the Massachusetts and New England experience

In February 1999, Massachusetts introduced expanded newborn screening for 20 rare metabolic disorders by MS/MS. Medium chain acyl-CoA dehydrogenase deficiency (MCADD) was mandated, in addition to 9 previously screened disorders, while the remaining 19 were offered as an optional pilot. Approximately 98% of parents have elected to participate in the optional program. Maine added MCAD in September 1999, and the optional disorders in July 2001. The other New England states are currently studying the benefits of adding additional testing. Expanded MS/MS screening in Massachusetts has thus far yielded a prevalence of approximately 1: 10,000 [200,000 screened--22 total cases--MCAD (7), VLCAD (1), SCAD (5), PA (2), 3-MCC (1), citrullinemia (1), ASA (1), argininemia (1), CPT11 (1) and 2 patients (argininemia and CPT11) with a severe neonatal presentation who died in the immediate newborn period]. All surviving patients have normal developmental outcomes so far. To evaluate the benefit of expanded newborn screening in the New England Region, the New England Consortium of Metabolic Centers has undertaken a prospective 3 year study, comparing the outcomes of patients identified by MS/MS with those diagnosed clinically. To date 22 screened patients (10 MCAD, 4 SCAD, 1 VLCAD, 1 CPT11, 13-MCC, 2 PA, 1 ASA, 1 citrullinemia and 1 argininemia) have been enrolled along with 24 clinically identified patients (7 MCAD, 1 SCAD, 1 VLCAD, 1 LCHAD, 1 argininemia, 1 tyrosinemia type 1, 6 PA, 3 GA 1, 2 GA 11, 1 MMA). Studies include: medical exam, neuropsychological assessment (Bayley Test and Stanford-Binet Test of Intelligence) and medical record review. Preliminary data suggest that the screened patients have an improved clinical outcome with fewer hospitalizations and so far, no neurological complications.     

Hyperhydroxyprolinaemia detected in newborn screening with tandem mass spectrometry

Hydroxyproline has the same integer molecular weight as leucine and isoleucine and is quantified with these by tandem mass spectrometry. An infant was diagnosed with hyperhydroxyprolinaemia following further evaluation of an elevated "leucine" level in newborn screening by tandem mass spectrometry.     

Pseudo-glutarylcarnitinaemia in medium-chain acyl-CoA dehydrogenase deficiency detected by tandem mass spectrometry newborn screening

Summary: As well as characteristic increases in C₈ carnitine, dried blood spot samples from 11 newborns with medium-chain acyl-CoA dehydrogenase deficiency detected by tandem mass spectrometry screening using butyl esters showed apparent increases in glutarylcarnitine (m/z 388 signals). In four of the newborns in which it was measured, apparent increases in malonylcarnitine (m/z 360) were also detected. It was shown that the apparent increases were caused by interfering acylcarnitines, putatively identified as hydroxyoctanoylcarnitine and hydroxydecanoylcarnitine, respectively, using alternative derivatives for tandem mass spectrometry. Levels of the two abnormal carnitines correlated with C₈ carnitine levels and normalized with repeat testing in 10 cases. These results indicated that the abnormal carnitines were significantly elevated only during periods of increased fatty acid catabolism, as may occur in the immediate postnatal period.     

Evaluation of 3-methylcrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry newborn screening

Summary: Since the addition of tandem mass spectrometry (MS/MS) to the North Carolina Newborn Screening Program, 20 infants with two consecutive elevated 3-hydroxyisovalerylcarnitine (C₅OH) levels have been evaluated for evidence of inborn errors of metabolism associated with this metabolite. Ten of these 20 infants had significant concentrations of both 3-hydroxyisovaleric acid and 3-methylcrotonylglycine in their urine, suggestive of 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency. Four of these 10 were infants whose abnormal metabolites were found to be of maternal origin. Of 8 patients with probable 3-MCC deficiency, 7 have been tested and found to have the enzyme deficiency confirmed in lymphoblasts or cultured fibroblasts; one of these 7 infants had only marginally decreased 3-MCC activity in lymphocytes but deficient 3-MCC in fibroblasts. We estimate the incidence of 3-MCC deficiency at 1:64000 live births in North Carolina. We conclude that MS/MS newborn screening will detect additional inborn errors of metabolism, such as 3-MCC deficiency, not traditionally associated with newborn screening. The evaluation of newborns with two abnormally elevated C₅OH levels on MS/MS newborn screening should include, at least, urine organic acid analysis by capillary GC-MS and a plasma acylcarnitine profile by MS/MS. Long-term follow-up is needed to determine the outcome of presymptomatically diagnosed patients with 3-MCC deficiency by MS/MS newborn screening.     

Structural characterization of toxic cyclic peptides from blue-green algae by tandem mass spectrometry.

Combined use of chemical degradation, derivatization, and tandem mass spectrometry for rapid structural characterization of toxic cyclic peptides from blue-green algae at the nanomole level is described. Previously, all blue-green algal toxins were thought to belong to a family of seven-residue cyclic peptides, having the general structure cyclo-D-Ala-L-Xaa-erythro-beta-methyl-D-isoaspartic acid-L-Yaa-Adda-D-isoglutamic acid-N-methyldehydroalanine, where Xaa and Yaa represent variable amino acids of the L configuration and Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid. Structural characterization of two additional toxins indicates that further variability can exist within this family of naturally occurring toxic cyclic peptides. Isoaspartic acid and dehydroalanine can substitute for beta-methylisoaspartic acid and N-methyldehydroalanine, respectively.     

液相串联质谱法定量检测高密度脂蛋白中脂质类化合物

目的本文旨在建立一种快速提取高密度脂蛋白(HDL)中脂质并结合液相串联质谱定性及快速定量检测的方法。方法采用酸化甲醇沉淀结合超声萃取的方法抽提脂质,高速离心去除蛋白颗粒,取上清液,直接采用液相串联质谱在多反应监测模式下进行定量分析。结果该分离提取脂质的方法简单迅速,采用多反应监测模式可同时定量监测30多种具有标准品的脂质成份,包括鞘氨醇类、鞘磷脂、神经酰胺和氧化型的卵磷脂。结论酸化甲醇超声抽提离心法可简便迅速地获得HDL中的脂质成份,结合液相串联质谱可对HDL中的多种脂质进行定量检测,该方法具有高效、重现性好等优点,适用于大量液体生物样本的分析。     

Protein identification using sequential ion/ion reactions and tandem mass spectrometry

A method for rapid sequencing of intact proteins simultaneously from the N and C termini (1-2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the N-Calpha bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 aa at both the N and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants.