Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry.
Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of "fluorous proteomics," whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.
Brittain
Scott M
SM
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Ficarro
Scott B
SB
Brock
Ansgar
A
Peters
Eric C
EC
D000345:Affinity Labels / Q000737:chemistry*; D002457:Cell Extracts / Q000032:analysis; D002458:Cell Fractionation; D005461:Fluorine / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008782:Methylurea Compounds / Q000494:pharmacology; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism
D000345:Affinity Labels; D002457:Cell Extracts; D008782:Methylurea Compounds; D010455:Peptides; D025801:Ubiquitin; C002249:O-methylisourea; D005461:Fluorine; D014357:Trypsin