The followings are a selection of experimental methods used by the Core
Facilities for Protein Structural Analysis, written up in a format suitable for
manuscript preparation. All users of the Core Facilities are welcome to reproduce the
relevant sections for their manuscript. Usually, less details are needed. More details
can be obtained upon request. If in doubt, or for specific applications where the
standard protocols may have been modified, please consult the Core Facilities
personnel.
Please note that an acknowledgement statement is required and the Core
Facilities should be notified of any accepted manuscript.
Acknowledgement
Proteomic mass spectrometry analyses were performed by the Core Facilities for
Protein Structural Analysis located at the Institute of Biological Chemistry, Academia
Sinica, supported by a National Science Council grant (NSC100-2325-B-001-029)
and the Academia Sinica.
MALDI MS and MS/MS Analysis
Protein spots or bands were subjected to concerted MALDI peptide mass
fingerprinting (PMF) and CID MS/MS analysis for protein identification using a
dedicated Q-Tof Ultima™ MALDI instrument (Micromass, Manchester, UK). The
instrument system was operated under MassLynx 4.0 and raw MS data were
processed for database searching using ProteinLynx Global Server 2.0.
For MALDI MS and MS/MS analysis, samples were premixed 1:1 with matrix
solution (5 mg/ml CHCA in 50% acetonitrile, 0.1% v/v TFA and 2% w/v ammonium
citrate) and spotted onto the 96 well format MALDI sample stage. Data directed
acquisition on the Q-TOF Ultima™ MALDI instrument was fully automated with
predefined probe motion pattern and peak intensity threshold for switching over from
MS survey scan to MS/MS, and from one MS/MS to another. Within each well, as
many parent ions meeting the predefined criteria (any peak within the m/z 800-3000
range with intensity above 10 count ± include/exclude list) will be selected for CID
MS/MS using argon as collision gas and a mass dependent ±5V rolling collision
energy until end of probe pattern was reached, starting from the most intense peak.
The LM and HM resolution of the quadrupole were both set at 10 to give a precursor
selection window of about 4 Da wide. For routine mode of operation, consideration
was given to acquiring sufficiently good quality MS and MS/MS spectra for both
PMF and MS/MS ions searching to increase the confident level of positive hits. The
instrument was externally calibrated to less than 5 ppm accuracy over the mass range
of m/z 800 - 3000 using a sodium iodide and PEG 200, 600, 1000 and 2000 mixtures
and further adjusted with Glu-Fibrinopeptide B as the near-point lock mass calibrant
during data processing. At a laser firing rate of 10 Hz, individual spectra from 5
second integration period acquired for each of the MS survey and MS/MS performed
were combined, smoothed, deisotoped (fast option) and centroided using the
Micromass ProteinLynx™ Global Server (PGS) 2.0 data processing software. The
combined PMF and MS/MS ion meta data were searched in concert against the
specified protein database within the PGS 2.0 workflow. Alternatively or additionally,
the PMF and individual MS/MS ion data can be output as Mascot-searchable .txt file
and .pkl files for independent searches against Swiss-Prot database using the Mascot
program.
nanoLC-MS/MS Analysis
The peptides mixtures were analyzed by online nanoflow liquid chromatography
tandem mass spectrometry (LC-MS/MS) on a nanoAcquity system (Waters, Milford,
MA) coupled to an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Scientific)
equipped with a PicoView nanospray interface (New Objective). Peptide mixtures
were loaded onto a 75-μm × 250-mm nanoACQUITY UPLC BEH130 column packed
with C18 resin (Waters, Milford USA) and were separated at a flow rate of 300 nl/min
using a linear gradient of 5 to 40% solvent B (95% acetonitrile with 0.1% formic acid)
in 30 min, followed by a sharp increase to 85% B in 1 min and held at 85% B for
another 10 min. Solvent A was 0.1% formic acid in water. The effluent from the
HPLC column was directly electrosprayed into the mass spectrometer. The LTQ
Orbitrap Velos instrument was operated in data-dependent mode to automatically
switch between full scan MS and MS/MS acquisition. Instrument control was through
Tune 2.6.0 and Xcalibur 2.1.
For the CID-MS/MS top20 method, full scan MS spectra (m/z 350–1600) were
acquired in the Orbitrap analyzer after accumulation to a target value of 1e6 in the
linear ion trap. Resolution in the Orbitrap system was set to R = 60,000 (all Orbitrap
system resolution values are given at m/z 400). The 20 most intense peptide ions with
charge states ≥2 were sequentially isolated to a target value of 5,000 and fragmented
in the high-pressure linear ion trap by low-energy CID with normalized collision
energy of 35%. The resulting fragment ions were scanned out in the low-pressure ion
trap at the normal scan rate and recorded with the secondary electron multipliers. Ion
selection threshold was 500 counts for MS/MS, and the maximum allowed ion
accumulation times were 500 ms for full scans and 100 ms for CID-MS/MS
measurements in the LTQ. An activation q = 0.25 and activation time of 10 ms were
used. Standard mass spectrometric conditions for all experiments were: spray voltage,
1.8 kV; no sheath and auxiliary gas flow; heated capillary temperature, 200°C;
predictive automatic gain control (AGC) enabled, and an S-lens RF level of 50%.
Data Analysis
All MS and MS/MS raw data were processed by Raw2MSM and searched
against a target protein sequence database using the Mascot Daemon 2.2 server.
Search criteria used were as follows: trypsin digestion; variable modifications set as
carbamidomethyl (Cys) and oxidation (Met); up to two missed cleavages allowed; and
mass accuracy of 10 ppm for the parent ion and 0.60 Da for the fragment ions.