PubMed Health⌕ Search

Biomedical subjects

A M Falick

Publications and source records attributed to A M Falick.

28 records · Page 2Linked to original sources
PubMed1991

Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins.

A combination of mass spectrometric (MS) techniques [electrospray MS, liquid secondary ion MS (LSIMS) and MS-MS] has been used for variant hemoglobin (Hb) detection and characterization. Electrospray MS allowed analysis of mixtures of intact globins giving simultaneously the molecular weights (accuracy 1-2 Da) and information about relative amounts of globins present. Currently, 14 Da is the minimum molecular weight difference required experimentally to accurately measure different species present in a mixture of 15-16 kDa proteins. Thus 80 and 79% of the known variants of alpha and beta chains, respectively, can be detected in mixtures with their normal counterparts, including Hb S (molecular weight difference = 30 Da). Abnormal hemoglobins detected were fractionated by C4 reversed-phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested by trypsin. The tryptic peptides were separated by C18 reversed-phase HPLC and analyzed by LSIMS to narrow down the mutation site to a single peptide. The mass measured in LSIMS frequently corresponded to a unique structure, thus giving the unequivocal identification of the mutation and its site. Where there was ambiguity, tandem MS on a Kratos Concept four-sector instrument was used for sequencing the abnormal peptide. The practical use of the methodologies presented is illustrated through analysis and identification of Hb G-San Jose, Hb Stanleyville II, Hb S and Hb Willamette.

Amino Acid Sequence↗
PubMed1990

Identification of a critical cysteine in EcoRI DNA methyltransferase by mass spectrometry.

EcoRI DNA methyltransferase (MTase) is rapidly inactivated by N-ethylmaleimide with concomitant incorporation of 2 mol of N-ethyl[2-3H]maleimide/mol of functional monomer. Preincubation of the enzyme with either S-adenosylmethionine or DNA reduces the rate of activity loss, whereas preincubation with DNA and the S-adenosylmethionine analog sinefungin completely protects the enzyme from inactivation. An endo proteinase Glu-C digest of N-ethyl[2-3H]maleimide-modified enzyme was prepared and separated by high pressure liquid chromatography. Modified and unmodified cysteine-containing peptides were located and identified by radioactivity, mass spectrometry, and tandem mass spectrometry. In the absence of any ligands, cysteines 25, 116, and 223 are modified by N-ethylmaleimide; in the presence of DNA and sinefungin, Cys-223 is essentially unmodified. Thus, N-ethylmaleimide modification of Cys-223 in EcoRI DNA MTase is responsible for the loss of enzyme activity. Cys-223 is preceded by Asn, and this (or Cys-Asn) occurs with high frequency in adenine and cytosine (N-4) DNA MTases. Direct involvement of cysteine in methyl transfer reactions to adenine N-6 and cytosine N-4 is supported by the similarity of the reactions catalyzed by adenine N-6 and cytosine N-4 DNA MTases, the frequent presence of Asn-flanking Cys, and the importance of Cys-223 to EcoRI MTase function.

Amino Acid Sequence↗
PubMed1990

N-terminus determination: FAD and NADP binding domain mapping of hog liver flavin-containing monooxygenase by tandem mass spectrometry.

Highly purified hog liver flavin-containing monooxygenase was sequentially denatured, reduced, carboxymethylated, and digested with endoproteinase Glu-C. The purified peptides were subjected to mass spectrometric analysis and the amino acid sequence of selected fragments was determined by tandem mass spectrometry. The amino acid sequence of the first 12 residues of the N-terminus was: Ac-Ala-Lys-Arg-Val-Ala-Ile-Val-Gly-Ala-Gly-Val-Ser-Gly. The amino acid sequence determined for another peptide was: Lys-Ser-Val-Leu-Val-Val-Gly-Met-Gly-Asn-Ser-Gly-Thr-Asp-Ile-Ala-Val-Glu. The results provide direct evidence for the structure of the N-terminal modification of the protein and for the existence of the FAD and NADP binding domains of Gly-X-Gly-X-X-Gly.

Acetylation↗
PubMed1990

Artifacts in four-sector tandem mass spectrometry.

Several types of artifacts were shown to be present in 4-sector tandem collision-induced dissociation (CID) mass spectra. In CID spectra of protonated peptides produced by liquid secondary-ion mass spectrometry (LSIMS), peaks corresponding to successive losses of matrix molecules from the precursor ion were observed. In addition, peaks corresponding to MH+ ions of smaller peptides that were also present in the sample/matrix mixture in greater abundance than the selected precursor ion were observed. Both of these types of artifact peaks were shown to originate from the 'peak-at-every-mass' chemical noise at the same nominal mass as that selected by the first 2 sectors (MS1). These noise ions are transmitted through to the collision cell and produce fragments that are analysed and detected in the next 2 sectors (MS2). A second, unrelated, kind of artifact was found to be due to decompositions in the second field-free region of MS2 in an EBEB geometry machine. These artifacts, which are detectable over only a very limited mass range when using a conventional single-point detector, can be present over a much greater mass range when an array detector is used and when the collision cell is floated above ground potential. A clear understanding of the origins of all peaks in a CID spectrum is important in order to have a firm foundation for interpretation, manual or computer-aided, of the spectra of unknown compounds.

Mass Spectrometry↗
PubMed1990

Tandem mass spectrometry in the clinical analysis of variant hemoglobins.

A combination of mass spectrometric techniques (electrospray mass spectrometry, liquid secondary-ion mass spectrometry (LSIMS), tandem mass spectrometry) has been used for variant hemoglobin detection and characterization. Electrospray mass spectrometry allowed analysis of mixtures of intact globins giving the molecular weights (accuracy 1-2 Da), and information about relative amounts of globins present, simultaneously. Abnormal hemoglobins detected in this way and by other means (screening, clinical symptoms) were fractionated by C-4 reverse phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested with trypsin. The tryptic peptides were separated by C-18 reverse phase HPLC and analysed by LSIMS to narrow down the mutation site to a single peptide. In some instances, the molecular weight of a variant peptide was sufficient to determine the mutation uniquely. When molecular weight information alone was insufficient to identify the mutation and its site, the peptide was sequenced by tandem mass spectrometry on a 4-sector instrument. In cases where more than one possible mutation site was present in the peptide and the mutation resulted in a change of only 1 Da in the peptide mass, the resolution and mass measurement accuracy of the 4-sector machine were essential in determining the correct sequence. The practical application of the methodologies presented is illustrated by the identification and analysis of Hb G-San Jose, Hb Willamette and D-Iran.

Hemoglobins, Abnormal↗
PubMed1990

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5.

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b5 were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from a Staphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariush et al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b5 were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2 +/- 2 and 9502.3 +/- 2 for bovine and rabbit trypsin-solubilized cytochrome b5, respectively. In both cases, these molecular weights correspond to a cytochrome b5 fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b5 was found to be 10,144.5 +/- 2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1-88) of Mr 10,146.1.

Amino Acid Sequence↗
PubMed1989

Derivatization of hydrophilic peptides for liquid secondary ion mass spectrometry at the picomole level.

A simple procedure for preparing alkyl and benzyl esters of peptides is described. The procedure can provide an increase in the secondary ion yield of a factor of 25 or more in the liquid secondary ion mass spectra of hydrophilic peptides. The procedure allows rapid in situ derivatization of, e.g., collected, lyophilized HPLC fractions. No sample transfers are required and excess reagents are easily removed. Mass spectrometry of such fractions is typically required to prepare a mass map of the peptides produced by proteolytic digestion of a protein. However, small hydrophilic peptides are often not detected because their low secondary ion yield. Relative yields of MH+ ions from peptides esterified with various alcohols are compared: methanol, 2-propanol, 1-butanol, 1-hexanol, 1-octanol, and benzyl alcohol. The best combination of ion yield and ease of reagent removal is obtained with 1-hexanol. The degree of improvement depends on the specific peptide; the greatest improvement is generally observed with the most hydrophilic peptides. The procedure does not affect side-chain amides. Partial derivatization is sometimes observed with peptides containing more than one carboxyl group. Hexylation is shown to have a leveling effect on the mass spectra of peptide mixtures, allowing detection of surface-inactive peptides in the presence of surface-active ones. Benzyl alcohol is useful for derivatizing peptides that are not retained or that elute very early from reverse-phase HPLC columns. The derivatives have longer retention times and greater uv molar absorptivity and are more easily detected by subsequent mass spectrometry than the underivatized peptides.

Esterification↗
PubMed1986

Cooled sample introduction probe for liquid secondary ionization mass spectrometry.

The design and performance of a cooled sample introduction probe for fast atom bombardment or other liquid secondary ionization mass spectrometric studies are described. Cooling relatively volatile matrix materials (sulfolane, thioglycerol, and tetraglyme, for example) in situ in the ion source can increase the duration of the sample spectrum by a factor of 10. Cooling also permits a much wider range of matrix materials to be used. As an example, the spectrum of the carbohydrate, peracetyl [Glu(beta 1----3)]7 Glucitol in tetraglyme matrix, is shown to give an excellent spectrum including cleavage peaks corresponding to losses of the first five sugar residues. The spectrum lasted approximately 10 times longer when the probe tip was cooled to 9 +/- 1 degrees C than when no cooling was used, corresponding to a 10-fold increase in integrated sensitivity.

Cold Temperature↗
PubMedDate not supplied

Identification of the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital.

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic known to be a relatively selective mechanism-based inactivator of rat liver cytochrome P450 2B1. Previous studies have demonstrated that such inactivation results in prosthetic heme destruction and irreversible drug-induced protein modification, events most likely triggered by P450 2B1-dependent oxidative activation of the olefinic pi-bond. However, the precise structure of the SB-modified heme and/or the protein site targeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [14C]SB in a functionally reconstituted system and structurally characterized it by electronic absorption spectroscopy and tandem collision-induced dissociation (CID), matrix-assisted laser desorption ionization on time of flight (MALDI-TOF), and liquid secondary ion mass spectrometry in the positive mode (+ LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX adduct. The [14C]SB-modified 2B1 protein has also been isolated from similar inactivation systems and subjected to lysyl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [14C]SB-modified 2B1 peptide was thus isolated, purified, electrotransferred onto a poly-(vinylidene) membrane, and identified by micro Edman degradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISLL293) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the distal helix I of P450 101, a region highly conserved through evolution, and which is known not only to flank the heme moiety but also to intimately contact the substrates. This finding thus suggests that SB-induced protein modification of P450 2B1 also occurs at the active site and, together with heme N-alkylation, contributes to the SB-induced mechanism-based inactivation of P450 2B1.

Amino Acid Sequence↗