Mass spectrometry in identification and characterization of biopolymers

Abstract

Advances in DNA sequencing has changed the focus of analytical biochemistry Advanced technology for determination of DNA sequences has become widely available in the last decade and have been used for sequencing cDNAs and entire genomes from a variety of organisms ranging from viruses to man. As a consequence, the amount o f DNA sequence information entered in publicly accessible databases has increased exponentially in the last decade. This growth has by far exceeded the growth of sequences entered in databases based on de novo protein sequencing. In addition to the genomic sequencing, large scale partial cDNA's sequencing has resulted in another set of data, the so-called expressed sequence tags EST's , containing stretches of sequence from a large number of genes from a variety of organisms. The genomic sequences only provides information of the potential of the selected microorganisms and cell types but does not re ect the actual situation at any given moment, i.e. which proteins are expressed and how are they modi ed. cDNA sequences or the incomplete EST's gives information on the actually expressed proteins, but no information on processing and secondary information. Therefore, studies of the proteins will never be obsolete, but the questions to address will be di erent. There is no doubt that the huge amount o f sequence information will be one of the most important sources to new knowledge about biological systems. However, e ective use of this information requires development of new analytical concepts by which data can be generated that matches the type of information in sequence databases.Mass spectrometry the ideal analytical technique in the post genome era Independently, but coinciding in time, mass spectrometric analysis has undergone an equally dramatic development. From being an analytical tool for analysis of small volatile molecules, new ionization methods, especially electrospray ionization ESI Fenn et al. 1989 and matrix assisted laser desorption ionization MALDI Karas and Hillenkamp 1988 , have increased the accessible mass range to include nearly all proteins. Mass accuracy and sensitivity h a ve been improved to a routinely achievable level Table 1 MALDI all modes 0.1 -1 fmoles 1 -10 fmoles 0.1 -1 pmoles nanoESI-quadrupole 10-50 fmoles 0.1 -1 pmoles 1-10 pmoles nanoESI-QTOF 1-10 fmoles 50-100 fmoles 0.5-5 pmolesMass spectrometry MS has been proven ideal for analysis of peptide and protein mixtures and partial or complete sequence information can be generated from the single components in such mixtures by the so-called MS MS techniques. In addition, MS is the ideal technique for analysis of post translational modi cations in proteins, thus being the perfect complement to DNA sequencing. Recent progress in nucleic acid and oligosaccharide analysis indicate that concepts similar for those described below for protein studies will also be applicable in studies of these biopolymers.Proteome analysis the next step after the genome Once a genome is sequenced then the next natural step is analy...

Truncated Semenogelin I Binds Zinc and Is Cleaved by Prostate‐Specific Antigen

“…The smaller rSgI 43 deviated slightly from the mass indicated by ProtParam (ie, +68 Da). This difference cannot be explained by mass inaccuracy, which was in the range of 0.01% (Roepstorff, 1999). Therefore, it cannot be excluded that an amino acid substitution occurred during synthesis of the recombinant protein, or that there were some minor posttranslational modifications such as oxidation.…”

Analysis of protein posttranslational modifications by mass spectrometry: With special reference to haemoglobin

“…Mass spectrometry (MS) has had a profound impact on addressing the structural problems involving many proteomes and other biopolymers (4). Much of the success of MS in biology has been largely due to the introduction of 'softionization' techniques like electrospray ionization mass spectrometry (ESI-MS) (5,6) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) (7,8), which can provide valuable information about the primary and higher orders of protein structures along with modifications without any disruption of covalent linkages (9). MS analysis has been particularly useful in identifying different variants of hemoglobin in conditions like thalassemias and hemoglobinopathies, which are inheritable genetic disorders.…”