Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

A screening method for the rapid detection of barbiturates in serum by means of tandem mass spectrometry.

A mass spectrometric screening method for barbiturates in serum was developed. Under electron impact conditions barbiturates fragment by loss of HNCO. A 'constant neutral loss' scan of 43 u provides, therefore, an indication of the presence of members of this toxicologically relevant class of compounds. Subsequent identification is possible either by 'daughter' or by 'parent ion' scans. The detection limit for propallylonal, buto- and phenobarbital was determined as better than 1 micrograms ml-1 serum.

[A screening method for rapid detection of barbiturates in serum using tandem mass spectrometry]. / Eine Screening-Methode zum schnellen Nachweis von Barbituraten im Serum mittels Tandem-Massenspektrometrie.

A mass spectrometric screening method for the detection of barbiturates in serum will be presented. Fragmentation of barbiturates under EI conditions leads to a loss of HNCO. Therefore, "neutral loss scans" of 43 u allow the indication of possibly existing representatives of this class of compounds. Identification can be achieved by "daughter" or "parent ion scans". Thus, 10 micrograms/ml apro-, pento- and phenobarbital could be detected in serum.