Friday, August 30, 2013

CHAMPS Antibody Sequencing Workflow

A couple of months ago, we have announced our CHAMPS antibody sequencing service. With the FREE "blind trials" we offered during the promotional period, we have received quite a few dataset from several users. The responses to the results we provided are remarkable.

Here is the general workflow we use to sequence an antibody.

We require the sample to be reduced with DTT, alkylated with iodoacetamide. Glycans need to be removed and heavy/light chains must be separated. Each chain then will be digested with six enzymes: AspN, chymotrypsin, GluC, LysC, pepsin and trypsin. MS/MS spectra is required to be acquired using LTQ-Orbitrap at high resolution with HCD fragmentation. In total, we require six LCMS runs per chain.

The data analysis starts off from PEAKS de novo sequencing. A list of high quality de novo sequences will be generated along with the positional confidence score for each amino acid. Then an in-house developed program will be used to assemble the de novo peptides into much longer sequences, protein contigs. In our experiments, the majority of assembled contigs had a length of 60~120 residues.

We blast the protein contigs in NCBI nr database to assemble the antibody template. We select a protein hit corresponding to the constant region and select the closest protein hit corresponding to the variable region. All the contigs will then be mapped to the template to get the first draft of the antibody sequence. In principle, we trust contigs in variable region and the template in constant region.

The draft sequence will be refined iteratively using PEAKS SPIDER homology search. During each iteration, we will examine insertion/deletion/mutation reported by SPIDER, residues with low peptide coverage, residues at the protein n-terminus and compare the sequence mass with protein intact mass, if available.

*Some content in this post is extracted from the ASMS 2013 poster "Whole Protein de novo Sequencing from MS/MS". You can find a web version of the poster here.

Wednesday, August 21, 2013

De Novo Assisted PTM "Blind Search" - PEAKS PTM

In PEAKS 6, we have introduced a new algorithm for PTM "blind search", PEAKS PTM. This algorithm can search for modified peptides with all 600+ PTMs in the Unimod database. To use this algorithm, simply select the option in the PEAKS Search dialog as shown below.

How is the "blind search" achieved? The secret is de novo sequencing. In usual PTM search algorithm, all possible modification forms of all database peptides satisfying the enzyme digestion rules are tried to match the spectra.

In PEAKS PTM, we only search for PTMs on the peptides when there is a tag match*. The algorithm also takes the PTM rarity into account to reduce the search space and false PTM assignment.

With PEAKS PTM algorithm selected, users only need to specify a very small number of variable PTMs in de novo and PEAKS DB to speed up the search and rely on PEAKS PTM to find other modifications presented in the sample.

*X. Han et al. PeaksPTM: Mass Spectrometry Based Identification of Peptides with Unspecified Modifications. JPR 2011, 10(7): 2930-2936.

Friday, August 9, 2013

de novo only peptides in PEAKS

One of the unique features in PEAKS is that it provides a list of de novo only peptides. What does it mean?

de novo only peptides are the de novo sequences derived from the spectra that do not a have confident database match. The following figure is a simple explanation. 

In PEAKS, de novo only peptides are listed in the de novo only tab in every PEAKS DB, PEAKS PTM, SPIDER and inChorus results. The actual de novo only peptides displayed in the list will be affected by the filters in the summary tab. Let's use PEAKS DB result below as an example.

The de novo only peptides are defined in the second row of the filters. The first part, TLC and ALC filters are the same as it is in the de novo result. It tells PEAKS what should be considered as a good de novo sequence. The second part, peptide -10lgP filter tells PEAKS what should not be considered as a confident database match. After the filters are applied, PEAKS will go through all the de novo sequences that passed the TLC and ALC filters. For each of such de novo sequence, PEAKS will look at the corresponding spectrum. If the spectrum does not produce any database matches that have a score higher than the de novo only peptide -10lgP filter, the de novo sequence of the spectrum will be considered as a de novo only peptide.

PEAKS does not stop at just providing a list of de novo only peptides, it also tries to associate them with the proteins. In the protein coverage view below, the gray bars represents the de novo only peptides that share at least 10 consecutive AAs with the protein sequence. This is particularly useful when looking for unexpected PTMs or glycosylation site.