Suspension-Cultured Plant Cells as a Tool to Analyze the Extracellular Proteome - Plant Proteomics: Methods and Protocols - page 402

3. Elute peptides at 300 nL/min fl ow rate by using a 40-min
linear gradient from 3 to 60 % ACN with 0.1 % FA.

4. Scan and fragment the peptides with a mass spectrometer
equipped with a nano-electrospray ion source ( see Note 10 ).
The three most intense precursor ions, ranging from 400 to
2,000 m/z , are scanned and measured in the mass spectrome-
ter at a 60,000 resolution at m/z 400, and the corresponding
fragment ions generated are measured in the mass spectrome-
ter after collision-induced dissociation (CID) fragmentation
using 35-40 % normalized collision energy.
5. Perform the database searches against NCBInr (non-redun-
dant) database ( ftp://ftp.ncbi.nih.gov/blast/db/FASTA/
nr.gz ) combining both MASCOT [ 34 ] and SEQUEST [ 35 ]
search engines implemented in Proteome Discoverer software
( see Note 10 ).
6. Use the following parameters for the searches: trypsin as the
proteolytic enzyme, allowing for one missed cleavage; carboxy-
amidomethyl cisteine as fi xed modifi cations; oxidation of
methionine as variable modifi cation.
7. Filter the resulting peptides to show the list of proteins identi-
fi ed with less than 1% false discovery rate. Consider positive
identifi cations only when two or more peptides are matched,
and their score is >20 for MASCOT and >2.5 for SEQUEST.

3.8 Peptide De Novo
Sequencing

1. Tryptic peptides generated from in-gel protein digestion are
analyzed by reverse-phase HPLC as described above
(Subheading 3.7 ). Both precursor and fragment ions are accu-
rately measured in the mass spectrometer and fragment ions
are generated by CID fragmentation.
2. Fragment spectra are de novo sequencing using Peaks Studio
v4.5 SP2 ( www.bioinformaticssolutions.com ). Parental and
fragment mass error tolerances are 0.01 and 0.02 Da, respec-
tively. Only those sequences with peptide scores ≥ 50 and qual-

ity values ≥ 0.75 and containing ≥ 4 amino acid strings with

confi dence 99.5 are considered as valid candidates. Fragment

spectra are also manually validated and the proposed sequences
searched using the BLASTP software [ 36 ].

≥

1. For sample in-gel rehydration [ 37 ], directly solubilize 500 μ g

3.9 Two-Dimensional
Gel Electrophoresis:
Isoelectrofocusing
Plus SDS-PAGE

of protein sample (pellet) in 250 μ L rehydration solution with

IPG buffer 3-10 ( see Note 24 ).
2. Select the Strip Holders corresponding to the IPG strip length
chosen for the experiment and 13 cm IPG strip pH 3-10 NL
( see Note 25 ), pipette 250

L of sample-containing rehydra-
tion solution into each strip holder base, and remove any large
bubbles.

μ