HybGFS: a hybrid method for genome-fingerprint scanning

Here, we present a hybrid method for genome-fingerprint scanning, known as HybGFS. This technique combines genome sequence-based peptide MS/MS ion searching with liquid-chromatography elution-time (LC-ET) prediction, to improve the reliability of identification. The hybrid method allows the simultaneous identification and mapping of proteins without a priori information about their coding sequences. The current study used standard LC-MS/MS data to query an in silico-generated six-reading-frame translation and the enzymatic digest of an entire genome. Used in conjunction with precursor/product ion-mass searching, the LC-ETs increased confidence in the peptide-identification process and reduced the number of false-positive matches. The power of this method was demonstrated using recombinant proteins from the Escherichia coli K12 strain.The novel hybrid method described in this study will be useful for the large-scale experimental confirmation of genome coding sequences, without the need for transcriptome-level expression analysis or costly MS database searching.Mass spectrometry (MS) is one of the major proteomics tools of the post-genomic era. Protein identification has traditionally been conducted by peptide mass fingerprinting (PMF) [1-3] or tandem MS (MS/MS) database searching and, while the former performs well with highly purified samples, the latter is the de facto standard for identifying proteins in complex samples. However, these approaches are only capable of detecting proteins already listed in databases. PMF implicitly assumes that all genes are annotated and that their complete protein sequences, including post-translational modifications, are known; however, in reality, most of these sequences are inadequately represented in existing databases. As alternative approaches, de novo peptide sequencing can be performed by the Edman degradation method [4], MS/MS [5,6], and controlled protein hydrolysis [7]. However, although amino-acid sequences can provide s