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A conceptually novel approach to protein sequencing involves the generation of ragged-end polypeptide chains followed by mass spectroscopic analysis of the resulting nested set of fragments. We report here on the synthesis and development of a volatile isothiocyanate (trifluoroethylisothiocyanate) that allows the identification of several consecutive residues starting with a few picomoles of peptide. The nested set of peptides is generated simply by adding equal aliquots of starting peptide each cycle and driving both the coupling and cleavage reactions to completion. No additional reagents are required to act as chain terminators and retention of the peptide terminal amine allows for subsequent modification with quaternary ammonium alkyl NHS esters to improve sensitivity. Complex washing procedures are not required each cycle, as reagents and by-products are efficiently removed under vacuum, eliminating extractive loss. Multiple peptide samples can be processed simultaneously, with each degradation cycle completed in 35-40 min. The inherent simplicity of the process should allow for easy automation and permit rapid processing of samples in parallel.

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We have developed a method to quantitatively attach quarternary ammonium fixed charge tags to the 5' or 3'NH 2 ends of DNA using N-hydroxysuccinimidyl ester chemistry. The chemical conditions for tagging were chosen so that tagging takes place exclusively on aliphatic NH 2 groups while base amino groups remain unmodified. The charge tagging chemistry was combined with a previously developed backbone alkylation procedure for phosphorothioate DNA. The efficiency of the detection in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of unmodified and modified DNA (phosphorothioate backbone, charge tagged, backbone alkylated, and charge tagged and backbone alkylated) was investigated using a series of different matrices. For α-cyano-4-hydroxycinnamic acid (a matrix, commonly used for the analysis of proteins, but which gives unsatisfactory results with unmodified DNA). For instance, the charge tagged and backbone alkylated DNA is detectable with a sensitivity and resolution comparable with that for peptides. The combination of charge tagging and backbone alkylation with the use of a suitable matrix improves the detectability of small oligonucleotides by MALDI by a factor greater than 100 compared to unmodified oligonucleotides. © 1997 by John Wiley & Sons, Ltd. 2 It has already found widespread use in peptide analysis whereas DNA analysis is still somewhat underdeveloped. The instability of DNA under the acidic conditions encountered during sample preparation and desorption and the high number of negative charges on the sugar phosphate backbone are major obstacles. Depurination, for instance, is a big problem and is observed at a rate of up to 2%. 3 It is thought that depurination takes place after base protonation and can be counteracted by replacing purines by their 7-deaza-purine analogues.4,5 A second possibility is the use of ribonucleotides where the 2'OH group inductively strengthens the N-glycosidic bond.6 A third possibility is the replacement of phosphate protons from the DNA backbone by alkyl groups.7 For quantitative and selective alkylation phosphorothioate bonds are required. Phosphorothioate substrates can replace regular substrates in most molecular biological procedures. This method has the added advantage of not only removing the acidity of the backbone but also the negative charges.In this paper we demonstrate the possibility of chemically modifying DNA to displace the negative charges from the sugar phosphate backbone and of attaching a single positive charge-tag prior to mass spectrometric analysis, thereby further simplifying the rather complex ionization of DNA during matrixassisted laser desorption. In this way DNA is made chemically more similar to proteins which are easily detected by MALDI. We also show the influence of the variation of matrix pK a values.

Summary In recent years, radiolabelled monoclonal antibodies have been evaluated for their use in the diagnosis and treatment of neoplastic disease. One isotope which has not been assessed for antibody targeting iS 32P, even though it has many favourable radiobiological characteristics and has been used clinically for the treatment of certain neoplastic disorders such as polycythaemia rubra vera. The main drawback so far in using 32P has been the absence of a general method for phosphorylating antibodies. We have now developed a novel process for the phosphorylation of immunoglobulins which is rapid, efficient and allows high specific activities to be achieved (>l10Ciyg-1). The Recently, however, a novel method for the covalent coupling of the strong chelating group diethylenetriaminepentaacetic acid (DTPA) to antibodies has been developed (Hnatowich et al., 1983a; Scheinberg et al., 1982;Paxton et al., 1985) which allows antibodies to be labelled with metallic radionuclides such as "'1In (Hnatowich et al., 1983a, b; Scheinberg et al., 1982;Paxton et al., 1985) and 99mTC (Lanteigne & Hnatowich, 1984).More recently another metallic radionuclide, 90Y, has been used to label DTPA-linked antibodies (Hnatowich et al., 1985) and promising radioimmunotherapeutic results have been reported (Order et al., 1986). This isotope is of considerable interest because of its pure f-ray particle emission, high emission energy, half-life and stable daughter products. However the use of 90Y does have disadvantages requiring the availability of a 90Sr-90Y generator and, in common with other chelated metallic radionuclides, there is a loss of 90Y from the antibody and large uptake by liver, kidneys, bone and bone marrow (Hnatowich et al., 1985).Another isotope with many of the ideal propeties of 90Y is 32p. Though having a longer half-life (14 days) than 90Y, this isotope has been used clinically in cancer therapy for many years (Today's drugs, 1967;Boye et al., 1984 advantageous for attacking tumours with low vascularity. Until now there has been no simple method for conjugating 32p to antibodies. In this communication we describe a simple, rapid procedure for phosphorylating antibodies which allows high specific activities to be achieved without any significant impairment of antibody function. Kemptide (Kemp et al., 1976), a heptapeptide substrate (Leu. Arg. Arg. Ala. Ser. Leu. Gly) for the cAMP-dependent protein kinase, is first covalently linked to the antibody and then phosphorylated with 32P-y-ATP and protein kinase. The antibody 32P-Kemptide conjugates show no impairment of antibody function, are stable in serum, and have a slow rate of clearance in mice (t,,2=2 days). Materials and methods ReagentsMonoclonal antibody secreting hybridomas were kindly provided by the following: OX7, Dr A