N-terminal sequence analysis of polypeptide at the picomole level.

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.     

Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate.

Polypeptides coupled with dimethylaminoazobenzene isothiocyanate through their amino groups to form dimethylaminoazobenzenethiocarbamoyl- (DABTC-)peptides can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). As little as 1 ng (2 pmol) of a DABTC-pentapeptide can be identified against a stable base-line with the signal-to-noise ratio of 10. The DABTC-peptides can also be recovered from the column, and their N-terminal amino acids (obtained by direct treatment with aqueous acid) and amino acid compositions and sequences can be all analysed at the picomole level. The power of this method is demonstrated by the complete separation and characterization of model peptides, peptide hormones and peptides derived from enzymic fragmentation of proteins. This new technique should provide a sensitive and efficient tool for peptide analysis at the nanogram level.     

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.     

High-performance liquid chromatography-based assay for the quantitation of protein maleylation at the picomole level

A new HPLC-based assay for the direct determination of the extent of maleylation of proteins is described. The rationale is founded on the HPLC detection of maleic acid that results from cleavage of maleyl lysyls by low pH treatment. The method, employing a C-18 reverse-phase column eluted isocratically with 0.01 M acetic acid, pH 5.4, and absorbance monitored at 210 nm, is sensitive (lower limit 1 pmol) and linear in the range 1 pmol-0.5 mumol and allows recovery of the native protein.     

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.     

Analysis of adenine nucleotides at the picomole level with 32P-phosphoenolpyruvate and pyruvate kinase.

A new sensitive method for adenine nucleotide analysis is described. The key reaction is the phosphorylation of ADP by [³²P]PEP in a reaction catalyzed by pyruvate kinase, with the extent of transfer of ³²P to ADP being determined by adsorbing the nucleotides onto charcoal. The nonadenine nucleoside diphosphates which also react in the pyruvate kinase reaction are corrected for by determining the ³²P retained in the nucleotide fraction after a second incubation with hexokinase and excess glucose. ATP is determined as ADP, after it is quantitatively converted by hexokinase in the presence of excess glucose. Similarly, AMP is analyzed by its conversion to ADP in an incubation with excess ATP and adenylate kinase. The sensitivity of the method for ADP and ATP is 0.05–0.5 pmoles while for AMP it is 5 pmoles.     

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.     

Comparative peptide mapping at the nanomole level

Comparison of highly similar proteins by peptide mapping at the 1-nmol level on cellulose-coated thin-layer sheets is described in detail. Maps were developed in three steps (a) a short chromatographic run in butanol:pyridine:acetic acid:H₂O (150:100:3:100), (b) electrophoresis at pH 3.5, and (c) rechromatography in butanol:pyridine:acetic acid:H₂O. The inclusion of step (a) greatly reduced streaking in the subsequent electrophoretic direction. Peptides were located by fluorescamine staining and could be eluted for composition or sequence analysis. To ensure complete comparison of the proteins two types of proteolytic digestion, a tryptic and a combined tryptic/thermolytic digestion, were necessary.     

Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay

An isoluminol assay is utilized for the detection of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described.     

Mass spectrometric analysis of cyanogen bromide fragments of integral membrane proteins at the picomole level: application to rhodopsin

Advances in time-of-flight mass spectrometry allow unit mass resolution of proteins and peptides up to about 6000 Da molecular weight. Identification of larger proteins and study of their posttranslational or experimental modifications by mass analysis is greatly enhanced by cleavage into smaller fragments. Most membrane proteins are difficult to mass analyze because of their high hydrophobicity, typical expression in low quantities, and because the detergents commonly used for solubilization may be deleterious to mass analysis. Cleavage with cyanogen bromide is beneficial for analysis of membrane proteins since the methionine cleavage sites are typically located in hydrophobic domains and cleavage at these points reduces the size of the hydrophobic fragments. Cyanogen bromide also gives high cleavage yields and introduces only volatile contaminants. Even after cleavage membrane proteins often contain fragments that are difficult to chromatograph. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is capable of analyzing complex mixtures without chromatography. We present a MALDI MS method that quickly and reliably identifies the cyanogen bromide fragments and posttranslational modifications of reduced and alkylated bovine rhodopsin from as little as 30 pmol of rhodopsin in detergent-solubilized retinal rod disk membranes, using 1-5 pmol of digest per sample. The amino acid sequences of some of the peptides in the digest were confirmed by post source decomposition MS analysis of the same samples. The method appears to be general and applicable to the analysis of membrane proteins and the protein composition of membrane preparations.     

High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide.

The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.     

Use of N-(7-dimethylamino-4-methylcoumarinyl)maleinimide for the detection of cysteine-containing peptides in peptide maps

A fluorescent reagent N-[7-dimethylamino-4-methylcoumarinyl]maleinimide (DACM), which reacts selectively with protein thiols, was used in the detection of cysteine-containing peptides in peptide maps. Direct staining of peptide maps of glutathione and tryptic digested α₁-antitrypsin resulted in the finding of one and four cysteine-containing spots, respectively. All other peptides could be visualized after the DACM staining, by the use of fluorescamine. Amino acid analysis of all peptides showed that only the DACM fluorescent spots contained cysteine residues.     

Determination of N-(9-( -D-ribofuranosyl)purin-6-ylcarbamoly)threonine at the picomole level in transfer-RNA

An assay has been developed for quantitation of the modified nucleoside, t⁶A, in tRNA at the pmole level. For tRNA from a variety of species, the content of t⁶A was found to be 0.18–0.25 mole %. These values lend support to the suggestion that t⁶A is located at the 3′-end of the anticodon in tRNA's whose codons begin with adenosine. Essentially no t⁶A was found in Mycoplasma sp. (Kid) tRNA which is deficient in many modified nucleosides. In the rat, no organ specific differences were found. The amount of t⁶A in Novikoff hepatoma tRNA was essentially the same as in tRNA from normal rat liver.     

Flow-injection resonance light scattering detection of proteins at the nanogram level.

A resonance light scattering (RLS) detection method for protein was developed, using a flow-injection system based on the enhancement of RLS signals from Biebrich scarlet (BS) by protein. The enhanced RLS intensities at 286.0 nm, in acidic aqueous medium, were proportional to the protein concentration over the range 0.005-18 microg/mL and 0.008-16 microg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively, with corresponding limits of detection (3sigma) of 5.00 ng/mL for HSA, and 7.80 ng/mL for BSA. The method was successfully applied to the quantification of total proteins in human serum samples.     

Nondestructive amino acid analysis at the picomole level of proline-containing peptides using aminopeptidase M and prolidase: application to peptides containing tyrosine sulfate.

A micromethod for nondestructive amino acid analysis was developed. Peptides were digested with amino-peptidase M followed by prolidase. The whole procedure was performed in a microvial for the autosampler of the analyzer. The resulting method has a working range from 10 to 600 pmol, at least, of sample taken into work. The usefulness of the method was demonstrated by its ability to determine the sulfation of tyrosine preceding proline in chicken gastrin.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.