Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level.
The post-genomic era and increased demands for broad proteome measurements have greatly increased the needs for protein identification. We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae. Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D(10)) had been added. The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum. The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein. Proteins can then often be unambiguously identified based on their accurately determined molecular mass and the additional constraint provided by number of Leu residues. The identities of proteins were further confirmed by repeating CIEF/FTICR measurements with samples that contain other isotopically labeled amino acid residues (e.g. His, Arg, Ile, Phe, Lys). A theoretical study of the amino acid composition (for a difference in the amino acid sequence) showed the constraints needed in order to identify the protein unambiguously. Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications.
Martinović
Suzana
S
Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratories, Richland, Washington 99352, USA.
Veenstra
Timothy D
TD
Anderson
Gordon A
GA
Pasa-Tolić
Ljiljana
L
Smith
Richard D
RD
D000596:Amino Acids / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D003470:Culture Media; D017357:Cyclotrons; D004926:Escherichia coli / Q000737:chemistry; D005583:Fourier Analysis; D005656:Fungal Proteins / Q000032:analysis*; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry
D000596:Amino Acids; D001426:Bacterial Proteins; D003470:Culture Media; D005656:Fungal Proteins; D020543:Proteome