Matsunaga, H., Santa, T., Hagiwara, K., Homma, H., Imai, K., Uzu, S., Nakashima, K., and Akiyama, S. (1995) Analytical Chemistry 67, 4276-4282. "Development of an Efficient Amino Acid Sequencing Method Using Fluorescent Edman Reagent 7-[(N,N-Dimethyl-amino)sulfonyl]-2,1,3-benzoxadiazol-4-yl Isothiocyanate {DBD-NCS}."
Synthesis, characterization, and use of DBD-NCS for high-sensitivity N-terminal sequencing. A detection limit of 50 fmol with a signal-to-noise ratio of 3 was observed during analysis of standard amino acid thiazolinone derivatives and known peptides. Chromatographic and Edman chemistry conditions were optimized.
Krause, I., Bockhardt, A., Neckermann, H., Henle, T., and Klostermeyer, H. (1995) Journal of Chromatography A 715, 67-79. "Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of dabsyl derivatives."
An automated derivatization/injection system is described for pre-column amino acid dabsyl analysis of protein hydrolyzates, amino acids, and biogenic amines arising from biological and food samples. Linearity ranged from 1.25 to 1250 pmol, detection limit from 0.12 to 0.52 pmol, and average repeatability from 1.3 to 3.1%. Triethylamine was shown to be an effective additive for optimizing resolution of amino acids and biogenic amines.
Lewisch, S. A. and Levine, R. L. (1995) Analytical Biochemistry 231, 440-446. "Determination of 2-Oxohistidine by Amino Acid Analysis."
2-Oxohistidine was stabilized during acid hydrolysis by addition of 40 mM DTT and maintaining the pH of the reconstitution/derivatization solution at 8 or less. This modified histidine residue could be detected in hydrolyzed samples at 1 pmol, in a background of 1700 pmol total amino acids, using pre-column derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate (AQC).
Weigt, C., Korte, H., Pogge von Strandmann, R., Hengstenberg, W., and Meyer, H. E. (1995) Journal of Chromatography A 712, 141-147. "Identification of phosphocysteine by electrospray mass spectrometry combined with Edman degradation."
During Edman sequencing, phosphocysteine is degraded primarily to cysteine and phosphate. Non-radioactive modification of phosphocysteine residues with ethanethiol provides a stable derivative, S-ethylcysteine, which can be identified in the PTH chromatogram. Sensitivity is in the low pmol range.
Licklider, l., Kuhr, W. G., Lacey, M. P., Keough, T., Purdon, M. P., and Takigiku, R. (1995) Analytical Chemistry 67, 4170-4177. "On-Line Microreactors/Capillary Electrophoresis/Mass Spectrometry for the Analysis of Proteins and Peptides."
Immobilized proteases (trypsin and carboxypeptidase Y) in a microcapillary reactor were used to generate peptide maps on low pmol quantities of substrates in picoliter to nanoliter volumes with minimal sampling handling. The digest was separated on-line via CE and fractions analyzed by ionspray MS. The entire process could be completed in less than 1 hour, and the use of "peptide fingerprinting" computer programs (e.g., MassMap) facilitated protein identification.
Walker, K. L., Chiu, R. W., Monnig, C. A., and Wilkins, C. L. (1995) Analytical Chemistry 67, 4197-4204. "Off-Line Coupling of Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry."
High-throughput, high-sensitivity peptide mapping can be achieved by directly depositing a portion of CE eluents on MALDI targets. Good MS spectra were obtained with as little as 25 fmol of protein and with a mixture of four proteins (150 fmol total). This procedure minimized sample handling, where the majority of losses occur.
Chakel, J. A., Apffel, J. A., and Hancock, W. S. (1995) LC-GC 13, 866-876. "Improvements in the Characterization of Glycopeptides and Glycoproteins Using Liquid-Phase Separations and Mass Spectrometry."
A short yet highly informative review outlining the multidimensional strategy of HPLC, CE, ESI-MS, and MALDI-MS collectively used to characterize glycosylated biological molecules.
Stults, J. T. (1995) Current Opinions in Structural Biology 5, 691-698. "Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)."
Up-to-date, lucid summary of advances in MALDI-MS sample preparation techniques, instrumentation, and analytical software. The recommended reading list contains 120 references with brief descriptions of articles of "outstanding interest".
Brown, R. S. and Lennon, J. J. (1995) Analytical Chemistry 67, 3990-3999. "Sequence-Specific Fragmentation of Matrix-Assisted Laser-Desorbed Protein/Peptide Ions."
A MALDI instrument using delayed pulsed ion extraction was operated in the linear mode to obtain sequence information. Incredibly, 80% of cytochrome C (12,360 Da) fragmented in a manner that allowed sequence assignment. (See Brown, R.S. and Lennon, J.J. (1995) Analytical Chemistry 67, 1998-2003 for a detailed report on the instrument.)
Solouki, T., Marto, J. A., White, F. M., Guan, S., and Marshall, A. G. (1995) Analytical Chemistry 67, 4139-4144. "Attomole Biomolecule Mass Analysis by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance."
The authors report the lowest detection limit--about 8 amol of substance P--claimed to date, obtained by FT-ICR/MALDI mass analysis. This was accomplished primarily by precise deposition of the analyte onto the probe tip with the aid of a microscope and multiple remeasurement of ions from a single laser shot. Prospects include MS analysis on single cell components.
He, L., Liang, L., and Lubman, D. M. (1995) Analytical Chemistry 67, 4127-4132. "Continuous-Flow MALDI Mass Spectrometry Using an Ion Trap/Reflectron Time-of-Flight Detector."
Ion trap storage in combination with reflectron MALDI-TOF MS allows direct introduction of solutions (via a continuous-flow probe) for analysis. Routine sensitivity is at the low pmol level for masses over 5000 Da using a resonance ejection technique to eliminate solvent and matrix ion peaks. Future considerations include interfaces to LC and CE systems.
Apffel, A., Fischer, S., Goldberg, G., Goodley, P. C., and Kuhlmann, F. E. (1995) Journal of Chromatography A 712, 177-190. "Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases."
Trifluoroacetic acid (TFA) causes "arcing" in many electrospray ionization instruments, ultimately suppressing analyte signal. A "TFA-fix" is described that increases signal/noise up to 100-fold by post-column addition of propionic acid:2-propanol (75:25, v/v) in a 1:2 proportion with the HPLC mobile phase. The authors report detection of less than 40 pmol protein from HPLC columns with 2.1 mm inner diameters when TFA is used as an ion-pairing agent in the mobile phase.
Patterson, D. H., Tarr, G. E., Regnier, F. E., and Martin, S. A. (1995) Analytical Chemistry 67, 3971-3978. "C-Terminal Ladder Sequencing via Matrix-Assisted Laser Desorption Mass Spectrometry Coupled with Carboxypeptidase Y Time-Dependent and Concentration-Dependent Digestions."
An experimental protocol employing parallel concentration-dependent in situ protease digestions on the MALDI target plate is described. This strategy effectively streamlines method development and consumes smaller amounts of substrate and enzyme than standard digestion protocols. Twenty-two peptides were subjected to this procedure, and 19 of them yielded some C-terminal sequence information.
Rosinke, B., Strupat, K., Hillenkamp, F., Rosenbusch, J., Dencher, N., Krüger, U., and Galla, H.-J. (1995) Journal of Mass Spectrometry 30, 1462-1468. "Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) of Membrane Proteins and Non-Covalent Complexes."
Three membrane proteins (porin, bacteriorhodopsin, and cholesterol esterase) were used as test substances to study the effects of detergents and matrices on MALDI-MS analysis of hydrophobic proteins. Seven detergents (3 non-ionic, 2 ionic, and 2 zwitterionic) were investigated for spectral sensitivity, background, and suppression. In general, better results were obtained with non-ionic detergents. Interestingly, a matrix of ferulic acid in tetrahydrofuran gave quaternary structural information for the three-subunit porin molecule.
Protein Characterization and Analysis
Patterson, S. D. and Aebersold, R. (1995) Electrophoresis 16, 1791-1814. "Mass spectrometric approaches for the identification of gel-separated proteins."
Comprehensive, current review on the state of MS as applied to protein identification of gel-separated samples with over 200 references. Topics include: biocompatible MS techniques, gel-separation methods, in situ fragmentation, electroelution, Edman and MS/MS sequencing, and computer program analysis of mass data to identify proteins.
Dogruel, D., Williams, P., and Nelson, R.W. (1995) Analytical Chemistry 67, 4343-4348. "Rapid Tryptic Mapping Using Enzymatically Active Mass Spectrometric Probe Tips."
A comparison between solution trypsin, agarose-immobilized trypsin, and trypsin covalently attached to a spectrometer probe tip for mapping lysozyme is reported. The probe-tip protocol was rapid and sensitive with no observable autolysis products. Other proteases should work as well, thus providing a "toolbox" of probe tips for peptide mapping.
Foret, F., Müller, O., Thorne, J. Götzinger, W., and Karger, B. L. (1995) Journal of Chromatography A 716, 157-166. "Analysis of protein fractions by micropreparative capillary isoelectric focusing and matrix-assisted laser desorption time-of-flight mass spectrometry."
An automated collection device for capillary isoelectric focusing analytes is described. Samples from a single run are collected at high precision allowing MALDI-TOF mass analysis of the entire electropherogram. In practice, it should now be possible to determine isoelectric points and molecular weights as a form of two-dimensional analysis on low pmol amounts of samples.
Houthaeve, T., Gausepohl, H., Mann, M., and Ashman, K. (1995) FEBS Letters 376, 91-94. "Automation of micro-preparation and enzymatic cleavage of gel electrophoretically separated proteins."
A processor capable of parallel handling of 32 samples is described. Gel pieces are washed, proteins are reduced and alkylated, proteolyzed, and the resultant peptide fragments extracted for HPLC, N-terminal sequencing, and MALDI-MS. This strategy provides a high-throughput system for analysis and identification (via computer search programs) of two-dimensional gel spots.
Gu, Q.-M. and Siu, C. J. (1995) International Journal of Peptide and Protein Research 46, 366-371. "Isolation and identification of C-terminal peptides of proteins."
A scheme is outlined that causes racemization of the C-terminal residue to the D-isomer, which is refractory to carboxypeptidases. The sample is N-trifluoroacetylated and reacted with acetic anhydride and pyridine. After removal of the trifluoroacetate group, the epimerized protein is digested with trypsin and then treated with several carboxypeptidases. When analyzed by RP-HPLC and compared to a non-racemized control, the C-terminal peptide can be readily identified.
Mannenberg, M., Lahm, H.-W., and Fountoulakis, M. (1995) Analytical Biochemistry 231, 349-353. "Quantification of Cysteine Residues Following Oxidation to Cysteic Acid in the Presence of Sodium Azide."
Continuing work from a previous Article Watch citation (December 1995) showing that acid hydrolysis in the presence of 0.2% (w/v) NaN3 results in quantitative conversion of cysteine and cystine residues to cysteic acid, which can then be readily analyzed. In comparison to performic acid hydrolysis, the NaN3 method can be performed in about half the time, gives slightly higher yields of cysteic acid, and does not appear to modify tyrosine residues.
Svoboda, M., Meister, W., and Vetter, W. (1995) Journal of Mass Spectrometry , 1562-1566. "A Method for Counting Disulfide Bridges in Small Proteins by Reduction with Mercaptoethanol and Electrospray Mass Spectrometry."
Rapid, simple method for determining disulfide bonds that relies on the formation of mixed disulfides with mercaptoethanol and a mass increase of 78. Potential to observe kinetics during reduction of samples containing multiple disulfide bonds. Successful analysis of samples containing 1, 2, 4, and 6 disulfide bonds and ranging from 1 to 23 kDa is demonstrated.
Return to the The ABRF Home Page