Precursor ion scans for the targeted detection of stable-isotope-labeled peptides.
Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large-scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse-labeled with a stable isotope by metabolic incorporation of 'heavy' amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple-quadrupole ion trap mass spectrometer. PIS-based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several-fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique.
Protein Analysis Facility, Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Switzerland. mara.colzani@unil.ch
Mara
M
Colzani
Willy V
WV
Bienvenut
Eveline
E
Faes
Manfredo
M
Quadroni
D045744:Cell Line, Tumor; D006801:Humans; D007477:Ions / Q000737:chemistry*; D007553:Isotope Labeling; D007554:Isotopes / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008545:Melanoma / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D007477:Ions; D007554:Isotopes; D010455:Peptides; D011506:Proteins