Quorum Sensing

3.6 Prepare the
Samples for MS
Analysis

1. When all the sample fractions are prepared, SpeedVac the
   eluted peptides to complete dryness. Store at 20 C until
   the day of MS analysis.


2. Dissolve samples in 10μl Buffer A prior to RP-HPLC-MS
   analysis.


3. Load each sample into the RP-HPLC that is of an Eksigent 2D
   nano LC system using a 80 mm fused silica emitter column of
   75 μm inner diameter (BGB Analytik) packed in-house with
   Magic C18 AQ 3μm resin (Michrom BioResources). Peptides
   are separated at a flow rate of 250 nl/min, against a gradient
   from 0.8% to 96.8% acetonitrile in 94 min.


4. Analyze the peptides on a LTQ-Orbitrap Velos mass spectrom-
   eter (Thermo Fisher Scientific) interfaced with a nanoelectros-
   pray source (seeNote 21) (or an alternative mass spectrometer
   with similar specifications regarding mass accuracy and sensi-
   tivity) by using the following parameters. Record survey scans
   recorded inm/zrange from 300 to 1700 with a resolution of
   30,000 atm/z400. Perform MS/MS experiments for the 20
   most intensive precursor ions as determined in the survey scan
   excluding unassigned charge states or singly charged ions from
   the MS/MS experiments in the linear ion trap.

3.7 Differential Data
Analysis

Several commercial software solutions are available for the analysis of

proteomics data [27]. We used an in-house pipeline for proteomics

searches and differential expression analysis developed by our colla-

borators [22]; it is based on the extraction of fragment ion mass

spectra from Thermo RAW files using msconvert (ProteoWizard,

version 3.0.3831), a search forB. cenocepaciaH111 matching pep-

tides with the powerful search engine MS-GF+ [22, 28], classifica-

tion of all identified peptides and filtering of only those that uniquely

identify one protein [29], and differential expression analysis with

DESeq [30] or similar software packages. To call a protein identified,

we applied the following criteria in Scaffold (peptide identification

probability 95%; protein identification probability 99%; at least two

peptides per protein) and the in-house pipeline (three PSMs for

peptides that unambiguously identify one protein). Differentially

expressed proteins were visualized in MA (“M” stands for log ratio

and “A” for mean average) plots, and proteins of particular interest

were independently validated with independent RT-PCR experi-

ments, knock-out mutants, or additional functional assays.

4 Notes

1. BDSF can be purchased from Sigma-Aldrich.


2. TCA is a strongly corrosive acid, which must be handled with
   care.

GeLC-MS/MS Proteomics to Identify Quorum Sensing Targets 199