Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2002
Laser ablation and ICP-MS operating conditions
The gels were ablated using the laser and the resulting plasma analysed using the
Platform ICP to give a scanning trace of total isotope concentration (TIC) as a
function of distance. The Platform ICP utilises a hexapole as the collision/reaction
cell, and so has the capabilities of eliminating Ar based molecular interferences. The
Platform ICP-MS conditions used are listed in Table 1.
Table 1: ICP-MS and laser conditions.
ICP-MS RF Power
1350 W
ICP-MS Cool Gas Flow
13.27 L·min-1
ICP-MS Auxilliary Gas Flow
0.82 L·min-1
ICP-MS Nebuliser Gas Flow
1.09 L·min-1
ICP-MS Helium Hexapole Gas Flow
10 mL·min-1
ICP-MS Hydrogen Hexapole Gas Flow
10 mL·min-1
ICP-MS Cone Voltage
70 V
ICP-MS Hex Exit Lens Voltage
400 V
ICP-MS Hex Bias Voltage
0.1 V
ICP-MS Ion Energy
2.0 V
ICP-MS Multiplier Voltage
400 V
Laser Frequency
200 Hz
Laser Power
100 %
Laser Spot Size
50 m
Laser Scan Speed
20 m·s-1
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2002
QTOF2 operating conditions
The mass spectrometer was operated in the positive ion mode with a source
temperature of 80 oC, a counter current gas flow rate of 40 l·h-1 and with a potential of
2600 V applied to the Nanospray continuous LC probe. All data were acquired with
the mass spectrometer operating in an automatic data dependent switching mode. The
instrument was calibrated with a fourth order calibration using selected ions from
Glu-fibrinopeptide-B.
Peptide separation protocol
The in-gel protein sample was automatically transferred to a 96-well plate and
dissected into 1 mm cubes, using the MassPrep station. The gel pieces were destained
by means of alternate ammonium bicarbonate and acetonitrile washes. The proteins
were then reduced and alkylated with the addition of dithiolthrionine and
iodoacetamide. Protein digestion was achieved with the addition of 25 l trypsin
solution (Promega, USA) at 6 ng·l-1 for 5 hours at 37 oC.
The resulting peptides were extracted in an aqueous solution containing 1 %
formic acid and 2 % acetonitrile. The peptides were lyophilised and reconstituted in 5
l of 1 % formic acid. The peptides were separated by means of a Micromass modular
capillary LC system connected directly to the Z-spray source of a Micromass Q-TOF2
mass spectrometer. Each sample was loaded on to a C18 pre-column (5 mm length,
320 m ID) at a flow rate of 30 l·min-1 and desalted for 3 min with a solution of 0.1
% formic acid. After desalting on the precolumn, the peptides were directed onto a
C18 Picofrit column (5 cm length, 75 m ID), and elution carried out using a solvent
gradient starting with 95 % solution A (95 % water, 5 % acetonitrile, 0.1 % formic
acid) and 5 % solution B (5 % water, 95 % acetonitrile and 0.1 % formic acid).
Following isocratic washing with this buffer, the peptides were eluted using a stepped
gradient to 80 % solution B.
Sequencing maps for OmpA
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2002
The masses and sequences peptide fragments produced by the trypsin digest of the
unknown protein sample were matched with the sequence of OmpA using
ProteinLynxTM Global server.
Table 2: Analysis of the peptides extracted after the in-gel trypsin digest of the
platinum-bound protein, showing (a) the results of the MS/MS sequencing of the
peptides produced from the trypsin digest and (b) the peptide fingerprint (bold)
mapped onto the whole sequence of the protein.
(a)
m/z
898.396
Charge
2
MW
1794.751
827.934
705.348
607.828
2
2
2
1653.825
1653.825
1213.612
528.257
458.282
436.777
409.734
2
2
2
2
1054.472
914.518
871.513
817.427
Sequence
(R)GMGESNPVTGNTCDNVK(Q) –
Carbamidomethylated
(K)LGYPITDDLDIYTR(L)
(K)LGYPITDDLDIYTR(L)
(R)AALIDCLAPDR(R) –
Carbamidomethylated
(K)DNTWYTGAK(L)
(K)AQGVQLTAK(L)
(R)RVEIEVK(G)
(R)LGGMVWR(A)
Residues
279-295
84-97
244-256
298-308
5-13
75-83
309-315
98-104
(b)
1
10
20
30
40
50
60
|
|
|
|
|
|
|
AAPKDNTMY TGAKLGWSQY HDTGFINNNG PTHENQLGAG AFGGYQVNPY VGFEMGYDWL GRMPYKGSVE
70
80
90
100
110
120
130
|
|
|
|
|
|
|
NGAYKAQGVQ LTAKLGYPIT DDLDIYTRLG GMVWRADTKS NVYGKNHDTG VSPVFAGGVE YAITPEIATR
140
150
160
170
180
190
200
|
|
|
|
|
|
|
LEYQWTNNIG DAHTIGTRPD NGMLSLGVSY RFGQGEAAPV VAPAPAPAPE VQTKHFTLKS DVLFNFNKAT
210
220
230
240
250
260
270
|
|
|
|
|
|
|
LKPEGQAALD QLYSQLSNLD PKDGSVVVLG YTDRIGSDAY NQGLSERRAQ SVVDYLISKG IPADKISARG
280
290
300
310
320
|
|
|
|
|
MGESNPVTGN TCDNVKQRAA LIDCLAPDRR VEIEVKGIKD VVTQPQA