Shotgun identification of protein modifications from protein complexes and lens tissue

Abstract

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co-and posttranslational modifications of proteins.

Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

“…In order to increase the chance of identifying modified sites, at least 80% sequence coverage is desired [47]. Use of other proteases with different cleavage specificity in addition to trypsin is a good way of increasing sequence coverage [14,49,50]. Jungblut and coworkers reported that they could achieve up to 80% sequence coverage for medium-quantity spots by PMM by employing two additional proteases, Asp-N and Glu-C, in addition to trypsin [47].…”

“…Because the effect was much higher from SEQUEST database search than from PROQUEST PMM search, only its effect on MS/MS data will be discussed. Oxidation of methionine is common modification occurring in proteins, but most of the modification detected by mass spectrometry is thought to come from adventitious oxidation of the residues when samples are exposed to acidic conditions and oxygen during protein processing and in-gel digestion [14]. Methionine can be oxidized to methionine-sulfoxide (ϩ16 Da) and further oxidized to methionine-sulfone (ϩ32 Da).…”

Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

“…The sample was reduced and alkylated using dithothreitol and iodoacetamide and then sequentially digested with endonuclease lyse-C (Roche Diagnostics, Indianapolis, IN) and soluble trypsin (Roche Diagnostics) (McCormack et al, 1997). The resulting peptide mixture was analyzed by MudPIT (Link et al, 1999;Washburn et al, 2001) with modifications described by McDonald et al (2002) and MacCoss et al (2002) (MacCoss et al, 2002;McDonald and Yates, 2002). Tandem mass spectra were searched against the latest version of the pompep database to which common contaminants such as keratin and trypsin were added (These sequence data were produced by the S. pombe Sequencing Group at the Sanger Centre and can be obtained from ftp://ftp.sanger.ac.uk/pub/yeast/Pombe/Protein_data/).…”

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines

“…To enhance the ability of separation and identification, especially toward the identification of protein modifications, MacCoss et al [73] have combined proteolytic cleavage of different enzymatic activities for the generation of overlapping peptides. To obtain more molecular level information for the intact proteins, direct coupling of multidimensional protein separation technologies [74 -76] with online trypsin reactor [77 -79] for real-time and effective digestion of resolved proteins will enable integrated top-down and bottom-up proteomics in a single analysis.…”