关键词： 酵母
TurboSEQUEST in the Proteomics Core
The Proteomics Core has purchased TurboSEQUEST which allows for faster search analysis times. This is primarily due to the ability to index for speed or size, databases to be used in the searching routine. We primarily index and use the non redundant database which we update on a regular basis. Moreover, subset databases can also be easily created from this database. Other databases can be also downloaded for searching (provided they are in the correct format).
Our version of TurboSEQUEST is web-based and can be accessed from any of our three terminals in the Proteomics Core. Regular users of the SWEHSC and the ACC who rely on TurboSequest will be given the URL to our server computer (with their own login and password) such that they can process/initiate their searches from their own PCs. Please note that the Proteomics Director reserves the right to pause searches so as to maintain a level of priority. In addition, you will need to obtain permission from the Proteomics Director and/or our personnel to initiate searches outside the Proteomics Core.
An LC-MS/MS example in the Proteomics Core
When presented with a protein gel band and you want to find its identity, what do you do?
The Proteomics Core has all the capabilities to identify your elusive protein band. Follow the steps below to see how a protein gel band is identified using LC-MS/MS.
(1) Take an example of a protein from a Coomassie stained gel brought to us from the laboratory of Dr. Anne E. Cress at the Arizona Cancer Center. This protein band migrated at a position on the gel indicating an average MW of 70 kDa.
(2) The band was excised and digested in-gel with trypsin as per our protocols.
(3) The extracted peptides were then separated on the TSP-4000 HPLC using a microbore C18 column (1.0 mm x 250 mm) at a flow rate of 10 uL/min. As the peptides were eluted off the column, they entered the LCQ mass spectrometer and were ionized. Their ionization in the form of ion current is plotted on the y-axis while the x-axis represents the time that the peptides eluted from the column.
(4) Let's look at what happens when a peptide is detected by the mass spectrometer and how that information is used to sequence a peptide. Take for instance the peak eluting at ~ 87 min above. The first thing that happens is that as the peptide is ionized, a mass spectrum is taken. The MS spectrumis shown below.
(5) Following the MS scan, each precusor peptide ion above a certain threshold is then isolated in the ion trap and subjected to collision-induced dissociation (CID) using a neutral gas. This results in dissociation of the peptide backbone into fragment ions. These fragment ions are then recorded. This is known as a tandem mass spectrum (MS/MS). The MS/MS spectra of peptides can be used to derive the sequence of the peptide. It should be noted that since we are dealing with typtic peptide ions (which posses a free N-terminus and either a lysine or arginie on the C-terminus), the precursor peptide ion (696.2 above) is assumed to be doubly charged (recall that the electrospray ionization source generates multiply charged molecules).
(6) Doubly charged precursor peptide ions undergoing CID fragment to generate sequence ions, where the charge can reside on either the N- or C-terminal end of the peptide. These are termed b and y ions, respectively. As shown below, the difference between successive b or y ions can give the sequence of the peptide.
The MS/MS spectrum of the precursor petptide ion at m/z 696.2 in the ion trap mass spectrometer gave the spectrum below. Determination of the b and y ions gives a partial sequence for that peptide.
(7) Undoubtedly to sequence peptides manually in the manner presented above would require a lot of time and effort. Software however, are available which sequence peptides in a highly automated manner. Moreover, these programs relate the peptides to their parent protein sequences, thus providing the identity of the protein. One such program that uses MS/MS spectra of peptides to identify proteins is SEQUEST, developed inthe laboratory of Dr. John R. Yates, III. The steps SEQUEST uses in protein identification are outlined below.
Candidate peptides from protein and nucleotide databases are chosen based on the precursor mass measurement (i.e.: the MS experiment) A ranking score system is applied to rank these candidate peptides The top 500 candidate peptides are kept A cross-correlation analysis is performed between the experimental MS/MS spectrum and the theoretical MS/MS spectrum of each of the 500 to candidate peptides An output file is given
For the experiment presented above, the output file is a web browser based spread sheet is shown below.
In reading this spread sheet, we see that in going across any one line, we obtain information on the total ion current signal (TIC) for each MS/MS spectrum that SEQUEST analyzed, its file name in terms of scan number, the prescursor charge state determined, the difference in mass between the experimental mass of the peptide and the best correlated peptide, the singly protonated mass of the peptide, the correlation value, the delta correlation, two scores, the experimental fragment ions vs the theoretical fragment ions for the peptide the program believes is the sequence of the peptide, the reference protein of the peptide and finally, the sequence of the peptide.
At the end of this spread sheet, the peptides are sorted and the best correlation-scored peptides are grouped by their origin protein. For the example, of the gel band investigated here, the summary is shown below.
The information that is given for the top scoring protein(s) include the protein ID/accession numbers (GenBank and Swiss-Prot) and the peptides that were sequenced via this correlation analysis. Each each protein identified is also linked to its sequence and to the peptides that were identified by SEQUEST. For this example, the top protein identified was a protein with GenBank accession number 106765. The information on this protein is shown below.
The peptides in red indicate the ones that were sequenced by this LC-MS/MS method. They represent 21.5% protein coverage in this 120 kDa protein. Moreover, they are exon regions of this alpha-6 integrin protein
Acquistion of a MALDI-TOF Mass Spectrometer in the Proteomics Core
The Proteomics Core has acquired a new matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) from Micromass. The new MALDI-LR is a dual linear/reflectron time-of-flight mass spectrometer ideal for protein mass fingerprining.
Below you will find an example of its utility for protein identification
(1) MALDI-TOF mass spectrum of an unknown protein
(2) Based on the mass measurement accuracy of the singly-charged peptides detected in the spectrum, protein identification is made
(3) A web browser interface shows the protein identified and the protein sequence coverge
