Identification of precursor forms of free prostate-specific antigen in serum of prostate cancer patients by immunosorption and mass spectrometry.

The structural features of the free prostate-specific antigen (F-PSA) present in human blood have not been clarified up to now, and it is, therefore, not known why F-PSA is not complexed by the protease inhibitors that are present in human blood in large amounts. This lack of information is mainly attributable to the low amount of F-PSA in serum, which makes the isolation and structural characterization very difficult, especially when only limited amounts of individual sera are available. It has now been demonstrated that F-PSA occurs as a mixture of different pro-PSA forms (zymogen forms) in the sera of prostate cancer patients, and that, in some of these sera, a form with the regular NH2 terminus of PSA is present as well. Among the five serum samples investigated, all contained the (-7), (-5), and (-4) pro-PSA forms, whereas the (-1) and (-2) forms were only present in three of them. These three samples also contained the form with the regular NH2 terminus. The (-3) and (-6) pro-PSA forms have not been detected thus far. The F-PSA has been isolated by immunosorption from the individual sera using streptavidin-coated magnetic beads. The pro-PSA forms were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after producing peptides by endoproteinase from Lysobacter enzymogenes digestion of the SDS-PAGE-separated F-PSA band. The structural identity of the (-7)pro-PSA form was further proven by sequencing of that particular peptide using electrospray ionization quadrupole time-of-flight mass spectrometry.

Automation of matrix-assisted laser desorption/ionization mass spectrometry using fuzzy logic feedback control.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a sensitive and versatile method for biomolecular analysis which has potential for high-throughput screening in many applications. To obtain mass spectra of optimal quality, however, laser fluence is continuously adjusted during data acquisition to be close to the threshold level of ion production, requiring a skilled operator and several minutes of acquisition time per sample. Using real-time fuzzy logic control of the laser fluence, we here demonstrate that the acquisition of MALDI spectra can be automated without reduction of data quality. The control algorithm evaluates signal intensity and mass resolution of the base peak. It then regulates the laser fluence to keep the ion signal intensity within the dynamic range of the data acquisition hardware while maintaining high mass resolution. This fuzzy logic control system allows unattended data acquisition using either static ion extraction or delayed ion extraction MALDI. Even for difficult samples such as femtomole-level peptide mixtures, no significant reduction in data quality is observed, as compared to manually obtained spectra. Automated analysis of 78 chromatographic fractions with high mass accuracy demonstrates the utility of the method. The control algorithm has been combined with other software modules to completely automate database identification of proteins by their peptide mass maps. The success of fuzzy logic in MALDI automation suggests wider uses of this technique in mass spectrometry.

Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots-many of them at subpicomole amounts-were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Sequence patterns produced by incomplete enzymatic digestion or one-step Edman degradation of peptide mixtures as probes for protein database searches.

Mass spectrometric peptide mapping of proteins isolated by polyacrylamide gel electrophoresis is a rapid method for identifying proteins in sequence databases. A majority of tryptic peptide maps were found to contain pairs of peptide ion peaks separated by the molecular weight of the lysyl or arginyl residue. These peaks originate from amino acid sequence patterns such as Lys-Lys where trypsin has cleaved C-terminals to either one of the lysines. The peptide mass and the pattern define an N- or C-terminal sequence tag. Searching sequence databases by such a sequence tag results in only a moderate number of matches and significantly reduces the number of database matches when used in combination with a peptide mass map. Two N- or C-terminal sequence tags alone unambiguously identify a protein in most cases. The technique discussed here is simple, does not require additional measurements, and increases the percentage of protein samples that can be identified by their mass maps alone. N-Terminal peptide sequence tags for database searching can also be generated by manual one-step Edman degradation of the unseparated peptide mixture.

Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.

Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Peptide sequence information derived by partial acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry.

Partial acid hydrolysis of purified peptides followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the resulting peptide mixture is a fast and simple procedure to confirm the identity of a peptide tentatively identified by its determined molecular weight. Often partial amino acid sequence information can be obtained directly. The sensitivity of the technique is in the high-femtomole to low-picomole range.

Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search.

Mass spectrometric peptide mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain peptide maps sufficient for protein identification was direct analysis of the extracted peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.

Improved mass accuracy in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides.

One problem of matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry is the moderate mass accuracy that typically can be obtained in routine applications, Here we report improved mass accuracy for peptides, even when low amounts and complex peptide mixtures are used. A new procedure for preparing matrix surfaces is used, and there is no need to mix the matrix with the sample or to add internal standards. Examples are shown with a mass accuracy better than 50 ppm in a peptide mixture. Peptide mapping as well as sequencing by creating "ragged ends" or "ladder sequencing" should benefit especially from the improved mass accuracy.

Synthesis and characterization of a 25-residue rubredoxin(II)-like metalloprotein and its valine-leucine mutant.

An iron-sulfur metalloprotein containing the 5-12 and 35-50 residues of Desulfovibrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val-5 and Glu-50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV-VIS spectrum of the reduced synthetic protein is very similar to that of native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (+/- 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre-requisite for protein formation. The Val-->Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.