Kristin Surmann
1,
*, Vera Laermann
2,
*, Petra Zimmann
3
,
Karlheinz Altendorf
2
, and Elke Hammer
1
1
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional
Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, 17475
Greifswald, Germany
2
Department of Biology/Chemistry, University Osnabrück, Barbarastraße 11, 49076
Osnabrück, Germany
3 University of Applied Sciences, Department A & L, Oldenburger Landstraße 62, 49090
Osnabrück, Germany
*Both authors contributed equally to this work.
1


Details on data acquisition for shotgun proteomics.

Presentation of protein identification results.

Details on data acquisition for MRM.

Details on method development and optimization for MRM.

Supporting Information Table 1. Composition of the minimal-medium.

Supporting Information Table 2. Incorporation rate of heavy standard peptides.

Supporting Information Table 3. Results from shotgun proteomics.

Supporting Information Table 4. Peptides of KdpABC and KdpDE identified by shotgun proteomics.

Supporting Information Table 5. Transitions used for identification and quantification by MRM.

Supporting Information Figure 1. CE optimization developed for two different peptides.

Supporting Information Table 6. Results of CE optimization.

Supporting Information Table 7. Fragment ion ratios of all transitions for cell lysate (0 mM K + ) spiked with 6 fmol or 60 fmol ‘heavy’ standard peptide.

Supporting Information Table 8. Fragment ion ratios of all transitions for cell lysate (115 mM K + ) spiked with 0.4 fmol or 0.8 fmol ‘heavy’ standard peptide.

Supporting Information Table 9. Proteotypic peptides used for quantification.

Supporting Information Table 10. Overview of the amount of proteins in the sample and the spiked-in peptides used for quantification.

Supporting Information Table 11. Absolute quantification results on peptide level for Kdp proteins after cultivation with 0 mM K + .

Supporting Information Table 12. Absolute quantification results on peptide level for Kdp proteins after cultivation with 115 mM K + .

Supporting Information Figure 2. The kdp regulon.
2

LC-column
LC-gradient
LC-Parameters
Solvent flow rate
Settings
Acclaim PepMap 100 reverse phase column
(3 µ m, 75 µ m i.d x 150 mm, LC Packings,
Dionex, Idstein, Germany)
0 min-1%ACN-35-1-36-5-245-25-305-60-
306-99-310-1-320-1
300 nL/min
MS-Parameters Settings
Mass range
Resolution m/z 300-2,000
60.000 at m/z 400
Name of peaklist-generating software and release version (number or date)
ReadW in Sorcerer built 4.04 (SageN
Research Inc., Milpitas, CA, USA) with default parameters
Name of the search engine and release version
(number or date)
Sequest (release 06/2011) in Sorcerer built
4.04 (SageN)
Enzyme specificity considered
# of missed cleavages permitted
Fully tryptic
Missed cleavages=0
Fixed modification(s) (including residue specificity)
Carbamidomethylation at cysteine
Variable modification(s) (including residue specificity)
Oxidation on methionine
Mass tolerance for precursor ions 10 ppm
Mass tolerance for fragment ions 1 Da
Name of database searched and release version/date
Swiss-Prot database rel. 57_10 limited to
E. coli K12 entries
Threshold score/E-value for accepting individual MS/MS Spectra
Peptide Teller false positive rate 1%
Software/method used to evaluate site assignment
No PTM reported
3

Information requested Reported
Accession number UniprotAccession (Table 1)
Number of unique (in terms of amino acid sequence) peptides identified
Table 1
% sequence coverage identified from MS/MS data or a list of sequences identified
% protein coverage in Supporting Information
Table 3, peptide sequences in Supporting
Information Table 4.
Additional information, such as a protein’s name, function, MW, pI, score, peptide sequences, etc.
MW, intensities are shown additionally in
Table 1; Supporting Information Table 3 contains the name of the proteins and their description, MW, pI, intensities; peptide sequences for selected proteins are provided in Supporting Information Table 4.
Single Peptide Protein IDs and PTMs Not reported in this manuscript.
4

LC gradient
LC-Parameters
Solvent flow rate
MS-Parameters
Settings
0 min-5%ACN-3-5-26-35-29-45-31-100-33-
100-36-0
300 nL/min
Settings
Resolution MS1 R=0.7 full width at half maximum
(FWHM), MS2 R= 2.5 FWHM
Dwell time
Cycle time
20 ms per transition
2.4 s/cycle
Workflow of method development, data analysis and absolute quantification of MRM data are explained in the text. MRM transitions are provided in Supporting Information Table 5, results from CE optimization are shown in Supporting Information Figure 1 and Supporting
Information Table 6. Fragment ion ratios are shown in Supporting Information Table 7 and 8.
Supporting Information Table 10 contains spike-in concentrations of heavy peptides.
5

A MRM approach allowing absolute quantification of the proteins of the Kdp system by adding known amounts of corresponding heavy labeled standard peptides to the samples was developed and optimized thoroughly. The general workflow is depicted in Figure 1.
Besides careful development of the MRM assay, also efficient cell lysis and complete digestion of the proteins are mandatory to obtain reproducibly reliable absolute amounts of proteins. Lysis efficiency was validated by light microscopy (Nikon Eclipse TS100 with 40x objective, Nikon GmbH, Düsseldorf, Germany). Equal numbers of bacteria were sampled by centrifugation as described. Four cell pellets were taken up in 300 µ L phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na
2
HPO
4
, 2 mM KH
2
PO
4
, pH 7.2-7.4) and counted with the help of a Thoma counting chamber (Poly-Optik GmbH, Bad Blankenburg,
Germany). Other four cell pellets were taken up in 300
µ
L UT-buffer and lyzed by ultrasonication as described. After centrifugation, remaining pellets were also investigated by microscopy after resuspension in 300
µ
L PBS. The resulting lysis efficiency by comparing numbers of cells from lyzed and non-lyzed pellets was determined to be >99%.
Digestion efficiency was tested with both types of samples (0 mM K
+
or 115 mM K
+
). Each
10 µ g protein were digested using trypsin as described in the materials and methods part.
Digested and non-digested samples were denatured (15 min, room temperature) and prepared for a NuPAGE Midi-Gel, 4-12%AA according to manufacturer’s instructions ( Novex Life
Technologies, Darmstadt, Germany). After silver staining, total staining intensity per lane was estimated to be less than 0.1% in comparison to that of the original protein lysate revealing a digestion efficiency of more than 99%.
As a first step of MRM method development, it was necessary to choose appropriate proteotypic peptides for each protein of the Kdp system. Proteotypic peptides are unique peptides of the protein of interest and detectable by MS [1]. Furthermore, tryptic peptides should not contain missed cleavage sites or methionine or cysteine, since those amino acids
6

are sensitive to chemical modifications which influence correct quantification. Proper peptides were primarily chosen from shotgun proteomic results (Supporting Information
Table 4), since peptides detected with good intensities in shotgun measurements showed also good signals in MRM analysis. If not enough peptides from shotgun analysis fulfilled the requirements [2], additional peptides derived from theoretical tryptic digestion of the amino acid sequences for each protein were added to the MRM acquisition list. For method development, all peptides matching these criteria were analyzed by MRM in a protein sample combining cells taken from both culture conditions. As many pairs of peptides and fragments
(transitions) as possible were measured and validated by their signal to noise ratio. For KdpA,
KdpB, and KdpC two peptides per protein were chosen and added as heavy labeled standard peptides containing
13
C- and
15
N-labeled arginine or lysine. For KdpD and KdpE, which were detected in very low concentrations in samples from cells grown in presence of 115 mM K
+
, more peptides were chosen to include one good performing peptide under these conditions.
Out of these 13 peptides finally chosen, eight were already detected in previous shotgun experiments and another five peptides were selected from theoretical considerations, which allowed advanced identification and quantification compared to shotgun analysis. Supporting
Information Table 5 contains all transitions, four for each peptide, which were monitored in the end.
Purity of the 13 heavy labeled standard peptides was determined by LTQ Orbitrap tandem mass spectrometry analysis of 100 fmol standard peptides digested with trypsin as described in materials and methods. Purity of the standard was determined by search against a complete
Swiss-Prot database (release 04/2012) to be >97% (other peptides belong to trypsin and keratin). Additional MRM scanning for the masses of light and heavy of four replicates using peak ratios (light/heavy) derived from the software Skyline resulted in 99.97% to 100% for incorporation rate of heavy isotopes for all peptides (Supporting Information Table 2).
7

To provide highest sensitivity and best quality of MRM measurements, the acquisition method was optimized for collision energy (CE) and LC gradient. The optimal value of CE which is applied during fragmentation of peptides isolated from MS depends on the size of the peptide and the instrument itself [3]. Therefore, adjustment was achieved by applying different CEs starting from factory defaults (depending on instrument and precursor m/z) for each transition and choosing the one which resulted in the highest intensity. For most peptides default electron voltage (eV) resulted in the highest peak intensities. However, for a few transitions an increase in the intensity of about 40% was detected after applying an adapted
CE. Supporting Information Figure 1 and Table 6 demonstrate the importance of CE optimization on the peptide intensity level. Furthermore, as in all LC-MS analyses it was necessary to adjust the ACN gradient in such a way that the best compromise in separation efficiency and peak height (data not shown) was achieved.
In order to ensure peptide ion identity in the complex samples, peptide retention time and fragmentation pattern of natural peptide and standard peptide had to be identical.
Fragmentation pattern was tested for each of both conditions by determination of the ratios of the fragment ions per precursor. They must be equal in the natural and the standard ion.
Differences might indicate the presence of interfering signals [4]. Due to varying concentrations of the Kdp proteins under both conditions (115 mM K
+
or 0 mM K
+
), fragment ion patterns were checked in both conditions by spike-in two different concentrations of heavy standard peptide each (Supporting Information Table 10). Results for the fragment ion ratios of all transitions from Supporting Information Table 5 of bacteria grown with 0 mM K
+ are listed in Supporting Information Table 7, those grown with 115 mM K
+
in Supporting
Information Table 8.
A further control of the quantification setup was achieved by creating standard curves for each heavy standard peptide (for linearity values see Supporting Information Table 9). All 13 peptides were added in four concentrations (Supporting Information Table 10) to natural
8

samples of E. coli lysate obtained from cells grown under limiting and non-limiting K
+
concentrations. Standard curves were developed from each five BRs for each concentration.
Most standard peptides showed a good linear regression throughout the whole concentration range (about two orders of magnitude) used (Supporting Information Table 9). Since the abundance of proteins in the sample with 115 mM K
+
is close to detection limit, corresponding natural peptides were not found in all samples. Also the slopes of peptides of one protein showed good correlation as exemplified for KdpC (Figure 2). The finally chosen proteotypic peptides which showed best linearity were therefore used for quantitative MRM analysis in this study are highlighted in Supporting Information Table 9 with sequences and molecular masses for the natural peptide (‘light’) and synthetic isotopically labeled standard
(‘heavy’).
For absolute quantification, samples were spiked with different concentrations of heavy standard peptides to identify the best concentration range for quantification. The aim was to reach an almost equal concentration of synthetic and natural peptides in order to lower the dynamic range in concentration and increase the accuracy of the measurement. As concentration varies between the two conditions of K
+
-supply, for each condition two different amounts were used for spike-in (Supporting Information Table 10).
Heavy peptides were spiked into the samples after protein determination but prior to digestion, in order to exclude as many technical variances as possible by treating sample and standard by the same protocol steps. When hydrolysis of the heavy standard is not required, e.g.
by using AQUA peptides in contrast to our peptides containing a tag which has to be removed, it is also common to add the heavy peptides after protease digestion to the peptides
[2, 5] but prior to C
18
purification to prevent inaccuracies due to possible sample loss.
Peak areas of natural sample peptides and corresponding heavy standard peptides eluting at the same RT were compared across replicates for each condition using the MRM setup described above. When more than one proteotypic peptide is used as spike-in, different
9

methods can be used for protein quantification like calculating median values in order to avoid outliers [6], average calculations from values derived from standard curves [7] or average calculation by prior weighting of peptides by the signal to noise ratios [8]. Final quantification of our data was achieved with the peptide showing a value of R 2 closest to one in the standard curves as described above. Of the two spike-in concentrations (Supporting
Information Table 10), the one which resulted in a heavy/light ratio closer to one was chosen for final calculations to avoid a too broad dynamic range. Such single-point calibration is possible when external calibration curves, developed with heavy standard peptides, show a good correlation (>0.9) [9] which was the case for all our standard peptides and spike-in concentrations used for quantification. Quantification results on the basis of individual peptides can be found in Supporting Information Table 11 (0 mM K
+
) and 12 (115 mM K
+
).
Furthermore, the advantage lies in the avoidance of technical variances, since the light natural peptide and the standard for quantification undergo digestion, acquisition and quantification in the same sample [9, 10].
References
[1] Kuster, B., Schirle, M., Mallick, P., Aebershold, R., Scoring proteomes with proteotypic peptide probes. Nat. Rev. Mol. Cell Biol. 2005 , 6 , 577-583.
[2] Kuzyk, M. A., Smith, D., Yang, J., Cross, T. J. et al., Multiple reaction monitoringbased, multiplexed, absolute quantification of 45 proteins in human plasma. Mol. Cell.
Proteomics 2009, 8 , 1860-1877.
[3] Maclean, B., Tozela, D. M., Abbatiello, S. E., Zhang, S. et al., Effect of collision energy optimization on the measurement of peptides by selected reaction monitoring (SRM) mass spectrometry. Anal. Chem . 2010, 82 , 10116-10124.
[4] Mani D. R., Abbatiello S. E., Carr S. A., Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics. BMC
Bioinformatics . 2012, 13 (Suppl 16): S9.
10

[5] Schmidt, C., Lenz, C., Grote, M., Lührmann, R., Urlaub, H., Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring. Anal. Chem. 2010, 82 , 2784-2796.
[6]
Maass, S., Sievers, S., Zühlke, D., Kuzinski, J. et al., Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal. Chem. 2011, 83 , 2677-2684.
[7] Langenfeld, E., Zanger, U. M., Jung, K., Meyer, H. E., Marcus, K., Mass spectrometrybased absolute quantification of microsomal cytochrome P450 2D6 in human liver.
Proteomics 2009, 9, 2313-2323.
[8] Werner, J. J., Ptak, A. C., Rahm, B. G., Zhang, S., Richardson, R. E., Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration. Environ. Microbiol . 2009, 11 , 2687-2697.
[9] Agger, S. A., Marney, L. C., Hoofnagle, A. N., Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple- reactionmonitoring mass spectrometry. Clin. Chem. 2010, 56 , 1804-1813.
[10] Tso, J., Dutta, S., Inamdar, S., Aga, D.S., Simultaneous analysis of free and conjugated estrogens, sulfonamides, and tetracyclines in runoff water and soils using solid-phase extraction and liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem.
2011, 59 , 2213-2222.
11

Supporting Information Table 1. Composition of the minimal-medium. After preparation of the medium, pH was set to pH 7.2.
K0 K115
46 mM 46 mM Na
2
HPO
4
× 2 H
2
O
NaH
2
PO
4
× H
2
O
MgSO
4
× 6 H
2
O
Tri-Na-Citrate
23 mM
0.4 mM
K
2
HPO
4
× 3 H
2
O
KH
2
PO
4
MgSO
4
× 6 H
2
O
Tri-Na-Citrate
23 mM
0.4 mM
Ammonium sulfate
FeSO
4
× 7 H
2
O
1 mM
8 mM
6
µ
M
Ammonium sulfate
FeSO
4
× 7 H
2
O
1 mM
8 mM
6
µ
M
12

Supporting Information Table 2. Incorporation rate of heavy standard peptides.
Purity of each 100 fmol heavy standard peptides was determined in four technical replicates scanning for the light and heavy masses using MRM. In some cases no peak for the light peptide was detected (na). CV – coefficient of variation
KdpA
KdpA
KdpB
KdpB
KdpC
KdpC
KdpD
KdpD
KdpD
KdpE
KdpE
KdpE
KdpE
Protein Peptide
ALGVSDR
LINDIPLPGTTGVER
ESGGDFASVTGGTR
GSLTTFSIANDVAK
YSQQPLVK
NLSVEQLTQLIAK
TYGLVVVEPGNLR
VYIAGQAER
LTLTASEEQAR
GLLEAATR
FSDVTVDLAAR
VFEAETLQR
QWSAVPVIVLSAR
Average ratio
‘light/heavy’
Standard deviation ratio
‘light/heavy’
CV [%]
0.00013
0.00030
0.00015
0.00023
0.00017
0.00020
0.00015 na na
0.00010
0.00010 na na
0.00006
0.00014
0.00010
0.00006
0.00012
0.00014
0.00007 na na na na na na
69
71
47 na
43
47
67
25 na na na na na
0.01
0.03
0.02
0.02
0.02
0.02
0.02 na na
0.01
0.01 na na
’light’ peptide
[%]
Purity of
‘heavy’ peptide
[%]
99.99
99.97
99.99
99.98
99.98
99.98
99.99
100.00
100.00
99.99
99.99
100.00
100.00
13

Supporting Information Table 3. Results from shotgun proteomics.
Results for identification and relative quantification of KdpABC subunits and KdpDE were obtained after data analysis via an Elucidator web interface (Ceiba Solutions, Boston, MA, USA) as average values from 3 BRs.
CV - coefficient of variation [%]
Primary Protein
Name
Protein
Description
Peptide
Count
Protein
Coverage
(%)
Protein
Mw [Da]
Protein
Pi
Protein
Teller
Probability
[K0 vs .
K115]
P-value
Intensity
[K0 vs .
1 [K115]
K115]
Intensity 2
[K0]
CV*
CV
[K115]
CV
[K0]
Factor 1 F
Ratio
Factor 1
P-value
Standard
Deviation
ATKA_ECOLI
ATKB_ECOLI
ATKC_ECOLI
KDPD_ECOLI
KDPE_ECOLI
Potassiumtransporting
ATPase A chain
GN=kdpA
Potassiumtransporting
ATPase B chain
GN=kdpB
Potassiumtransporting
ATPase C chain
GN=kdpC
Sensor protein kdpD
GN=kdpD
KDP operon transcriptional regulatory protein kdpE
GN=kdpE
1
16
3
8
1
3
29
26
12
4
59189.1
72199.2
20267.16
98718.40
25362.2
7.00
7.58
6.59
6.13
5.99
0.97
1
1
1
1
2.1
20.4
75.8
15.7
15.5
0.191 30557
3.22E-07 864294
2.09E-07
3.26E-08
3.80E-05
31136
67967
6172
63232
17670000
2361414
1069496
95607
69.8
106.7
114.3
102.6
94.5
105.2
21.3
53.3
56.7
142.9
45.9
32.2
32.9
29.1
7.2
1.71
26.11
26.95
30.55
109.61
0.261
0.007
0.007
0.005
0.00
32714.70
9885353.00
1367783.88
583373.25
56527.86
CV* coefficient of variation between the two conditions
14

Supporting Information Table 4. Peptides of KdpABC and KdpDE identified by shotgun proteomics. Most peptides mentioned in this table were identified in shotgun proteomics (3 BRs). Peptides highlighted in grey were added additionally to this list from theoretical tryptic digestion of the protein sequences as not enough peptides from shotgun analysis could be used. They could be acquired in MRM mode. Peptides were crossed out when they did not fulfill requirements for proteotypic peptides as e.g.
they contained cysteine or methionine. Peptides in bold letters represent the peptides ordered as heavy standards.
Swiss-Prot
ID
Primary Protein
Name
Gene name Protein Description Peptide Name
P03959
P03959
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03960
P03961
P03961
P03961
P21865
P21865
P21865
P21865
P21865
P21865
P21865
P21865
P21865
P21865
P21866
P21866
P21866
P21866
P21866
ATKA_ECOLI
ATKA_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKB_ECOLI
ATKC_ECOLI
ATKC_ECOLI
ATKC_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPD_ECOLI
KDPE_ECOLI
KDPE_ECOLI
KDPE_ECOLI
KDPE_ECOLI
KDPE_ECOLI kdpA kdpA kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpB kdpC kdpC kdpC kdpD kdpD kdpD kdpD kdpD kdpD kdpD kdpD kdpD kdpD kdpE kdpE kdpE kdpE kdpE
Potassium-transporting ATPase
A chain
Potassium-transporting ATPase
A chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
B chain
Potassium-transporting ATPase
C chain
Potassium-transporting ATPase
C chain
Potassium-transporting ATPase
C chain
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
Sensor proteinkdpD
KDP operon transcriptional regulatory protein kdpE
KDP operon transcriptional regulatory protein kdpE
KDP operon transcriptional regulatory protein kdpE
KDP operon transcriptional regulatory protein kdpE
KDP operon transcriptional regulatory protein kdpE
LINDIPLPGTTGVER
ALGVSDR
SIVILAK
GSLTTFSIANDVAK
VLGVIALK
DVQSLHATFVPFTAQSR
TLADAAQLASLADETPEGR
MLGANVIATSGR
MSGINIDNR
ESGGDFASVTGGTR
EAGNMVDLDSNPTK
MIAMVEGAQR
AVEAAGDVDVLLLDK
LNPQAQWR
LTAAAIAAEAGVDDFLAEATPEAK
LIEVVHIGK
TVMITGDNR
VIDLLLTVCGLV
NLSVEQLTQLIAK
YSQQPLVK
PSATAEMPYNPQASGGSNLAVSNPELDK
VYIAGQAER
LAQELGAETATLSDPAEEK
QLMIPEQQR
VTLPQQTAPELEEFHEDM
IAPDLDQVLVALDEPPAR
LVNNLLDMAR
HISEFDLDAALAR
TWAMLAEAQR
TYGLVVVEPGNLR
LTLTASEEQAR
LLAVLLNNAGK
VFEAETLQR
GLLEAATR
QWSAVPVIVLSAR
FSDVTVDLAAR
15

Supporting Information Table 5. Transitions used for identification and quantification by MRM. List of sequences and fragments used for quantification of natural peptides (‘light‘) with the help of standard peptides containing
13
C and
15
N (‘heavy’).
Peptide
ALGVSDR
ALGVSDR
ALGVSDR
ALGVSDR
ESGGDFASVTGGTR
ESGGDFASVTGGTR
ESGGDFASVTGGTR
ESGGDFASVTGGTR
‘light‘
Precursor
Ion
359.198
359.198
359.198
359.198
670.807
670.807
670.807
670.807
FSDVTVDLAAR
FSDVTVDLAAR
FSDVTVDLAAR
FSDVTVDLAAR
GLLEAATR
GLLEAATR
GLLEAATR
GLLEAATR
GSLTTFSIANDVAK
597.312
597.312
597.312
597.312
415.740
415.740
415.740
415.740
712.375
GSLTTFSIANDVAK
GSLTTFSIANDVAK
GSLTTFSIANDVAK
LINDIPLPGTTGVER
LINDIPLPGTTGVER
LINDIPLPGTTGVER
LINDIPLPGTTGVER
LTLTASEEQAR
LTLTASEEQAR
LTLTASEEQAR
LTLTASEEQAR
NLSVEQLTQLIAK
NLSVEQLTQLIAK
NLSVEQLTQLIAK
NLSVEQLTQLIAK
QWSAVPVIVLSAR
712.375
712.375
712.375
797.944
797.944
797.944
797.944
609.820
609.820
609.820
609.820
728.922
728.922
728.922
728.922
713.414
QWSAVPVIVLSAR
QWSAVPVIVLSAR
QWSAVPVIVLSAR
TYGLVVVEPGNLR
TYGLVVVEPGNLR
TYGLVVVEPGNLR
TYGLVVVEPGNLR
VFEAETLQR
VFEAETLQR
VFEAETLQR
VFEAETLQR
VYIAGQAER
VYIAGQAER
VYIAGQAER
VYIAGQAER
YSQQPLVK
YSQQPLVK
YSQQPLVK
YSQQPLVK
713.414
713.414
713.414
708.896
708.896
708.896
708.896
546.788
546.788
546.788
546.788
503.769
503.769
503.769
503.769
481.769
481.769
481.769
481.769
846.432
717.389
646.352
517.309
907.463
744.400
631.316
560.279
799.467
712.435
584.377
456.318
891.417
790.369
719.332
1043.610
914.567
786.508
673.424
1111.683
953.614
854.546
545.341
883.500
784.431
685.363
556.320
Product
Ion
646.352
533.268
476.246
377.178
895.463
748.395
677.358
491.257
959.516
745.420
644.373
545.304
660.368
Peptide
ALGVSDR
ALGVSDR
ALGVSDR
ALGVSDR
‘heavy‘
Precursor
Ion
364.202
364.202
364.202
364.202
ESGGDFASVTGGTR
ESGGDFASVTGGTR
ESGGDFASVTGGTR
ESGGDFASVTGGTR
FSDVTVDLAAR
FSDVTVDLAAR
FSDVTVDLAAR
FSDVTVDLAAR
GLLEAATR
547.283
418.241
GLLEAATR
GLLEAATR
347.204 GLLEAATR
1065.558 GSLTTFSIANDVAK
675.812
675.812
675.812
675.812
602.316
602.316
602.316
602.316
420.744
420.744
420.744
420.744
716.382
964.510
817.441
GSLTTFSIANDVAK
GSLTTFSIANDVAK
617.325 GSLTTFSIANDVAK
1139.642 LINDIPLPGTTGVER
1026.558 LINDIPLPGTTGVER
929.505 LINDIPLPGTTGVER
816.421
1004.501
LINDIPLPGTTGVER
LTLTASEEQAR
716.382
716.382
716.382
802.948
802.948
802.948
802.948
614.824
LTLTASEEQAR
LTLTASEEQAR
LTLTASEEQAR
NLSVEQLTQLIAK
NLSVEQLTQLIAK
NLSVEQLTQLIAK
NLSVEQLTQLIAK
QWSAVPVIVLSAR
QWSAVPVIVLSAR
QWSAVPVIVLSAR
QWSAVPVIVLSAR
TYGLVVVEPGNLR
TYGLVVVEPGNLR
TYGLVVVEPGNLR
TYGLVVVEPGNLR
614.824
614.824
614.824
732.929
732.929
732.929
732.929
718.418
718.418
718.418
718.418
713.900
713.900
713.900
713.900
VFEAETLQR
VFEAETLQR
VFEAETLQR
VFEAETLQR
VYIAGQAER
VYIAGQAER
VYIAGQAER
VYIAGQAER
YSQQPLVK
YSQQPLVK
YSQQPLVK
YSQQPLVK
551.792
551.792
551.792
551.792
508.774
508.774
508.774
508.774
485.776
485.776
485.776
485.776
557.292
428.249
357.212
1073.572
972.524
825.456
625.340
1149.650
1036.566
939.513
826.429
1014.509
901.425
800.377
729.340
1051.624
922.581
794.523
681.439
1121.692
Product
Ion
656.360
543.276
486.255
387.186
905.471
758.403
687.366
501.266
969.524
755.429
654.381
555.312
670.376
963.622
864.554
555.349
893.508
794.439
695.371
566.328
856.440
727.397
656.360
527.318
917.471
754.408
641.324
570.287
807.481
720.449
592.391
464.332
16

Supporting Information Figure 1. CE optimization developed for two different peptides.
The red graph shows factory defaults for CE. On the left, peptide TYGLVVVEPGNLR gives the highest intensities and peak areas (bar plot) when applying factory defaults. For peptide
FSDVTVDLAAR, CE was adjusted after optimization as a higher CE resulted in higher peak areas.
17

Supporting Information Table 6. Results of CE optimization. Values of CE optimization for each peptide are given in %. For most peptides there was no significant change in signal intensity, for others, e.g
. ESGGDFASVTGGTR, peak areas increased by about 41% after optimization.
Peptide
LINDIPLPGTTGVER
ALGVSDR
ESGGDFASVTGGTR
GSLTTFSIANDVAK
NLSVEQLTQLIAK
YSQQPLVK
VYIAGQAER
TYGLVVVEPGNLR
LTLTASEEQAR
VFEAETLQR
GLLEAATR
QWSAVPVIVLSAR
FSDVTVDLAAR
Increase in area by applying CE different from factory default [%]
25
8
11
0
18
3
38
41
6
20
7
9
27
18

Supporting Information Table 7. Fragment ion ratios of all transitions for cell lysate (0 mM K + ) spiked with 6 fmol or 60 fmol ‘heavy’ standard peptide.
Transitions from Supporting Information
Table 5 were used to acquire fragment ion peak areas of each precursor which were related to the peak area of the most intense fragment peak (lines highlighted in grey). As fragment ion ratios should be same between sample peptide and internal standard, both ratios were again compared. Ion ratios which exceeded a fold change of 2 were colored (ratio <0.5 blue, ratio >2 red). Peptides used for quantification were colored in green. Frag. - Fragment
Protein Peptide Frag. ion
RT
60 fmol
Frag. ratio
'light' 60 fmol
Frag. ratio
'heavy'
60 fmol
Ratio
60 fmol
RT 6 fmol
Frag. ratio
'light' 6 fmol
Frag. ratio
'heavy' 6 fmol
Ratio
6 fmol
KdpA
KdpB
KdpC
KdpD
LINDIPLPGTTGVER
ALGVSDR
ESGGDFASVTGGTR
GSLTTFSIANDVAK
NLSVEQLTQLIAK
YSQQPLVK
VYIAGQAER
TYGLVVVEPGNLR
LTLTASEEQAR y9 y11 y8 y10 y6 y4 y3 y5 y9 y8 y5 y7 y6 y10 y9 y8 y8 y7 y6 y9 y5 y7 y6 y4 y8 y5 y6 y7 y6 y5 y7 y8 y9 y7 y8 y6
18.03
18.03
18.03
18.03
6.85
6.85
6.85
6.85
11.66
11.66
11.66
11.66
17.18
17.18
17.18
17.18
22.28
22.28
22.28
22.28
9.03
9.03
9.03
9.03
8.76
8.76
8.76
8.76
17.7
17.7
17.7
17.7
10.91
10.91
10.91
10.91
0.01
0.02
0.17
1.00
0.03
0.06
0.18
1.00
0.35
0.39
0.61
1.00
0.60
0.63
0.82
1.00
0.45
0.48
0.82
1.00
0.13
0.58
0.80
1.00
0.04
0.52
0.63
1.00
0.57
0.71
0.76
1.00
0.47
0.94
0.93
1.00
0.01
0.02
0.17
1.00
0.03
0.08
0.18
1.00
0.34
0.37
0.59
1.00
0.53
0.64
0.85
1.00
0.50
0.50
1.07
1.00
0.11
0.58
0.78
1.00
0.05
0.55
0.66
1.00
0.54
0.74
0.75
1.00
0.47
0.93
0.95
1.00
0.98
0.90
1.00
1.00
0.92
1.50
0.98
1.00
0.98
0.94
0.97
1.00
0.89
1.01
1.05
1.00
1.11
1.04
1.31
1.00
0.81
1.00
0.98
1.00
1.21
1.06
1.05
1.00
0.96
1.04
0.98
1.00
1.01
0.99
1.02
1.00
17.0
6
22.1
2
22.1
2
17.0
6
17.0
6
17.0
6
22.1
2
22.1
2
8.82
8.82
8.82
8.82
8.56
8.56
8.56
8.56
17.6
4
17.6
4
17.6
4
17.6
4
10.7
7
10.7
7
10.7
7
10.7
7
17.9
1
17.9
1
17.9
1
17.9
1
6.5
6.5
6.5
6.5
11.5
2
11.5
2
11.5
2
11.5
2
0.01
0.02
0.16
1.00
0.04
0.04
0.15
1.00
0.34
0.39
0.59
1.00
0.60
0.67
0.94
1.00
0.41
0.41
0.79
1.00
0.13
0.61
0.79
1.00
0.06
0.51
0.57
1.00
0.58
0.73
0.78
1.00
0.50
1.00
1.00
1.17
0.01
0.01
0.18
1.00
0.04
0.12
0.12
1.00
0.31
0.34
0.64
1.00
0.50
0.52
0.72
1.00
0.43
0.36
0.95
1.00
0.12
0.56
0.58
1.00
0.05
0.57
0.64
1.00
0.56
0.74
0.79
1.00
0.61
0.91
1.00
0.99
0.61
0.75
1.12
1.00
1.01
2.85
0.80
1.00
0.92
0.87
1.09
1.00
0.83
0.77
0.77
1.00
1.06
0.87
1.20
1.00
0.97
1.02
1.01
1.00
1.22
0.91
1.00
0.84
0.89
0.92
0.73
1.00
0.81
1.13
1.12
1.00
19

KdpE
VFEAETLQR
GLLEAATR
QWSAVPVIVLSAR
FSDVTVDLAAR y5 y7 y5 y9 y5 y11 y8 y6 y9 y4 y5 y6 y7 y3 y6 y4
1.00
0.14
0.06
0.31
1.00
0.29
0.34
0.35
1.00
0.16
0.31
0.46
1.00
0.30
0.82
1.25
12.27
21.3
21.3
21.3
21.3
15.2
15.2
15.2
15.2
12.33
12.33
12.33
12.33
12.27
12.27
12.27
1.00
0.09
0.15
0.24
1.00
0.35
0.38
0.40
1.00
0.20
0.22
0.46
1.00
0.27
0.79
0.80 0.64
1.00
0.65
2.48
0.76
1.00
1.22
1.09
1.15
1.00
1.24
0.72
0.99
1.00
0.88
0.97
12.1
3
21.2
21.2
21.2
21.2
15.1
2
15.1
2
15.1
2
15.1
2
12.2
12.2
12.2
12.2
12.1
3
12.1
3
12.1
3
1.00
0.10
0.08
0.30
1.00
0.42
0.50
0.57
1.00
0.22
0.26
0.50
1.00
0.25
0.67
1.05 0.77
1.00
1.15
3.16
0.92
1.00
1.30
0.73
0.62
1.00
0.93
0.93
0.96
1.00
0.89
1.26
1.00
0.12
0.27
0.27
1.00
0.54
0.36
0.35
1.00
0.21
0.24
0.48
1.00
0.23
0.84
0.81
20

Supporting Information Table 8. Fragment ion ratios of all transitions for cell lysate
(115 mM K + ) spiked with 0.4 fmol or 0.8 fmol ‘heavy’ standard peptide.
Transitions from
Supporting Information Table 5 were used to acquire fragment ion peak areas of each precursor which were related to the peak area of the most intense fragment peak (lines highlighted in grey). As fragment ion ratios should be same between sample peptide and internal standard, both ratios were again compared. Ion ratios which exceeded a fold change of 2 were colored (ratio <0.5 blue, ratio >2 red). Peptides used for quantification were colored in green. Peaks which could not be distinguished from noise or were not present at all are marked ‘na’. Frag. - Fragment
Protein
KdpA
KdpB
KdpC
KdpD
KdpE
Peptide
LINDIPLPGTTGVER
ALGVSDR
ESGGDFASVTGGTR
GSLTTFSIANDVAK
NLSVEQLTQLIAK
YSQQPLVK
VYIAGQAER
TYGLVVVEPGNLR
LTLTASEEQAR
VFEAETLQR
GLLEAATR
QWSAVPVIVLSAR
FSDVTVDLAAR
Frag. ion y5 y6 y7 y6 y7 y8 y5 y8 y7 y9 y8 y6 y5 y7 y6 y4 y8 y9 y8 y5 y7 y6 y9 y10 y9 y11 y8 y10 y6 y5 y4 y3 y5 y4 y3 y9 y8 y11 y5 y9 y7 y6 y5 y4 y6 y7 y6 y9 y7 y8 y6 y5
RT 0.8 fmol
16.88
22.04
22.04
22.04
22.04
8.52
8.52
8.52
8.52
8.19
8.19
8.19
8.19
17.41
17.41
17.41
17.41
17.71
17.71
17.71
17.71 na na na na
11.19
11.19
11.19
11.19
16.88
16.88
16.88 na na na
21.10
21.10
21.10
21.10
12.71
12.71
12.71
12.71
10.38
10.38
10.38
10.38
11.88
11.88
11.88
11.88 na
0.98
1.09
1.00
0.53
0.32
0.70
1.00
1.00 na na na na
1.12
0.62
3.38
1.00
0.03
Frag. ratio
'light'
0.8 fmol
0.55
0.10
0.49
1.00 na na na na
0.44
0.33
0.92
11.00
2.26 na
0.27 na na na na na na na na na na na
0.38
1.56
1.51
1.00
0.95
0.26
0.91
1.00 na
Ratio
0.8 fmol
0.09
0.90 na na
0.21 na
3.11
RT
0.4 fmol
17.46
17.46
0.58 17.46
1.00 17.46 na na na na na na
0.53 11.15
1.51 11.15
0.57 11.15
0.09 11.15
16.67
16.67
16.67
1.00 16.67 na na na na na na na
0.10
0.44
0.12
1.00
3.13
0.48 na
8.52
8.52
8.52
8.52
8.03
8.03
1.00 na na na na
0.29
0.32
0.34
1.00
0.08
0.59
1.00
0.97
2.13
8.03
8.03
17.15
17.15
0.60
0.72
1.00
0.16
0.63
1.17 17.15
1.00 17.15
1.00
1.64
1.27 10.36 22.71
0.47 10.36 41.35
0.55 10.36 44.00
1.00 10.36 1.00
0.25 11.84 0.57
Frag. ratio
'light'
0.4 fmol
0.28
0.10
0.31
1.00 na na na na
0.86
0.74
1.00
1.00
5.04
0.37
0.41
0.96 11.84
0.43 11.84
1.00 11.84 na na na na na na na na na na na na na na na na
20.81
20.81
20.81
20.81
19.16
19.16
19.16
19.16
0.16
1.00
0.13
0.16
0.14
0.21
0.17
1.00
0.17
0.55
1.00 na na na na
0.48
0.65
1.00
0.52
0.68
0.82
1.00
1.00
0.35
0.49
0.66
1.00
0.11
0.28
0.41
1.00
0.10
Frag. ratio
'heavy'
0.8 fmol
0.05
0.09
0.29
1.00 na na na na
0.23
0.50
0.52
1.00
0.48
0.81
0.84 na na na
0.06
0.43
0.49
1.00 na na na na
0.48
0.73
0.83
1.00
0.24
0.26
0.39
1.00 na
0.41
0.02
0.02
0.02
1.00
0.43
1.75
1.03
3.31
1.27
0.58
1.00
6.23
1.19
1.00
0.26
0.62
1.00
0.10
0.53
0.60
1.00 na na na na
1.55
1.23
1.29
1.00
0.82
2.88
1.94
1.00 na na na na
0.36
1.00 na na na na
1.00
1.00
1.70
5.92 na na na na
0.76
0.42
1.00
0.97
0.74
1.00
0.68
1.00 na na na na
0.45
0.40
0.43
1.00
0.25
Frag. ratio
'heavy'
0.4 fmol
0.23
0.29
0.61
1.00 na na na na
0.31
0.19
0.62
1.00
0.51
0.19
0.25 na na na
0.16
1.00
0.22
0.92 na na na na
0.52
0.82
0.96
1.00
0.24
0.29
0.57
1.00 na
Ratio
0.4 fmol
21

Supporting Information Table 9. Proteotypic peptides used for quantification.
List of sequences used for quantification of natural peptides (‘light’) with the help of standard peptides containing
13
C- and
15
N-labeled Arg and Lys (‘heavy’). In addition, for each standard peptide four different concentrations were spiked into sample background and peak area as well as linearity between concentration and peak area were compared (n=5). Sequences of the peptides chosen for quantification of the protein are highlighted in grey and written in bold letters.
Protein Peptide
Mass 'light'
[g/mol]
Mass 'heavy'
[g/mol]
R 2
KdpA LINDIPLPGTTGVER 1593.887
ALGVSDR*
KdpB GSLTTFSIANDVAK
716.396
1422.750
KdpC
ESGGDFASVTGGTR
YSQQPLVK
1339.615
961.538
NLSVEQLTQLIAK 1455.844
KdpD TYGLVVVEPGNLR* 1415.792
1603.895
726.405
1430.764
1349.623
969.552
1463.858
1425.800
0.9997
0.9950
1.0000
0.9998
0.9997
0.9997
0.9997
VYIAGQAER
LTLTASEEQAR*
KdpE QWSAVPVIVLSAR*
GLLEAATR
1005.539
1217.640
1424.829
829.480
1015.547
1227.648
1434.837
839.489
0.9994
0.9997
1.0000
0.9999
VFEAETLQR 1091.576 1101.584 0.9999
FSDVTVDLAAR* 1192.623 1202.632 1.0000
* peptides which were not found in shotgun analysis but were chosen from theoretical tryptic digestion.
22

Supporting Information Table 10. Overview of the amount of proteins in the sample and the spiked-in peptides used for quantification. As amounts of peptides of Kdp(F)ABC complex were much higher in K
+
-limiting medium, higher amounts of standard peptides were added to the samples.
Concentration of K + [mM]
0
0
115
115
Spike-in ‘heavy’ peptide [fmol/µg protein]
100
10
1
0.5
‘Heavy’ peptide on column
[fmol]
60
6
0.8
0.4
Protein amount on column [µg]
0.6
0.6
0.8
0.8
23

BR5 0.07
BR1 0.07
BR2 0.06
BR3 0.04
BR4 0.04
BR5 0.05
BR1 0.15
BR2 0.16
BR3 0.09
BR4 0.08
BR5 0.11
BR1 0.03
BR2 0.05
BR3 0.03
BR4 0.03
BR5 0.03
BR1 0.03
BR2 0.02
BR3 0.03
BR4 0.03
BR5 0.02
BR1 0.11
BR2 0.07
BR3 0.09
BR4 0.11
BR5 0.11
BR1 0.03
BR2 0.04
BR3 0.02
BR4 0.02
BR5 0.02
BR1 0.88
BR2 0.68
BR3 0.45
BR4 0.44
BR5 0.55
BR1 0.23
BR2 0.21
BR3 0.18
BR4 0.15
BR5 0.17
BR1 1.23
BR2 0.89
BR3 0.58
BR4 0.71
BR5 0.90
BR1 0.56
BR2 0.48
BR3 0.38
BR4 0.40
BR5 0.44
BR1 1.09
BR2 1.04
BR3 0.56
BR4 0.57
BR5 0.73
BR1 0.66
BR2 0.66
BR3 0.33
BR4 0.36
BR5 0.43
BR1 0.10
BR2 0.07
BR3 0.05
BR4 0.06
Supporting Information Table 11. Absolute quantification results on peptide level for Kdp proteins after cultivation with 0 mM K + . Total amount of sample protein was 0.6
µ g. Peptides for protein quantification due to ratio close to 1 and best R 2 are highlighted in green, outliers removed from calculation in gray. CV - coefficient of variance, SD - standard deviation.
Protein
KdpA
KdpB
KdpC
KdpD
KdpE
Peptide
LINDIPLPGTTGVER
ALGVSDR
ESGGDFASVTGGTR
GSLTTFSIANDVAK
NLSVEQLTQLIAK
YSQQPLVK
VYIAGQAER
TYGLVVVEPGNLR
LTLTASEEQAR
VFEAETLQR
GLLEAATR
QWSAVPVIVLSAR
FSDVTVDLAAR
BR
Ratio
'light'/
'heavy'
2.39
11.39
7.40
9.45
11.44
10.57
2.59
4.04
1.50
2.48
1.82
3.11
4.54
2.52
2.79
2.73
2.77
2.39
2.56
2.81
42.96
10.38
6.52
5.20
6.34
6.96
7.08
5.85
4.13
4.34
5.18
15.06
15.53
8.65
8.09
10.67
89.57
55.60
48.39
37.98
40.33
43.80
109.09
103.86
56.44
57.00
73.16
65.96
65.83
33.42
35.91
60 fmol 'heavy' peptide
Natural Average peptide per µg amount per
SD
[fmol] sample
[fmol/µg] peptide
[fmol]
87.77
68.20
44.80
43.86
55.15
59.96 18.39
23.44
21.30
17.87
15.32
17.34
122.70
88.71
58.05
70.62
19.05
85.93
3.26
24.42
45.22
79.91
48.82
7.00
25.23
15.98
7.08
5.32
11.60
3.14
2.58
10.05
2.49
1.96
1.20
3.51
0.81
0.20
1.69
0.98
CV
[%]
31
17
28
15
32
33
28
23
30
26
8
17
39
Ratio
'light'/
'heavy'
2.20
10.44
10.69
6.78
9.45
7.53
2.36
2.04
1.46
2.43
2.12
3.50
4.26
2.29
3.64
3.25
3.00
2.83
1.98
2.79
43.07
13.09
11.30
5.50
5.67
7.35
8.35
6.25
3.43
4.83
5.45
16.99
13.91
7.14
8.90
11.21
79.59
55.74
39.76
22.93
39.50
32.14
141.25
140.24
51.44
57.49
67.72
70.33
98.49
27.50
34.34
6 fmol 'heavy' peptide
Natural Average peptide per µg amount per
SD
[fmol] sample
[fmol/µg] peptide
[fmol]
105.42
106.00
37.70
47.59
57.04
70.75 32.64
CV
[%]
46
17.23
0.01
9.60
14.13
12.63
139.21
144.47
57.76
68.12
10.72
97.83
6.59
40.95
61
42
38.01
91.63
54.75
12.06
45.21
29.37
32
49
54
8.58
5.66
11.63
3.39
2.56
8.98
2.08
3.43
1.82
3.93
0.72
0.44
1.75
0.38
40
32
34
21
17
19
18
0.32
0.30
0.28
0.20
0.28
0.22
1.04
1.07
0.68
0.95
0.75
0.24
0.20
0.15
0.24
0.21
0.71
0.89
1.12
0.35
0.43
0.23
0.36
0.74
0.83
0.63
0.34
0.48
0.54
1.70
1.39
7.03
9.85
2.75
3.43
4.31
1.31
1.13
0.55
0.57
3.95
3.21
14.12
14.02
5.14
5.75
6.77
1.26
13.92
14.45
5.78
6.81
7.96
5.57
3.98
2.29
10.54
10.60
3.77
4.76
5.70
1.72
0.00
0.96
1.41
24

Supporting Information Table 12. Absolute quantification results on peptide level for Kdp proteins after cultivation with 115 mM K + . Peptides for protein quantification due to ratio close to 1 and best R 2 are highlighted in green, outliers removed from calculation in gray. Not all peptides could be detected under this condition; those are not included in this table. CV - coefficient of variation, SD
- standard deviation.
Protein
KdpA LINDIPLPGTTGVER
KdpB
KdpC
KdpD
KdpE
Peptide
ESGGDFASVTGGTR
GSLTTFSIANDVAK
YSQQPLVK
VYIAGQAER
TYGLVVVEPGNLR
LTLTASEEQAR
VFEAETLQR
QWSAVPVIVLSAR
BR
Ratio
'light'/
'heavy'
BR1 0.08
BR2 0.21
BR3 0.50
BR4 0.18
BR5 0.34
BR1 0.13
BR2 0.31
BR3 0.37
BR4 0.25
BR5 0.36
BR1 0.26
BR2 0.60
BR3 0.33
BR4 0.50
BR5 0.48
BR1 0.44
BR2 0.77
BR3 0.44
BR4 0.72
BR5 0.40
BR1 0.15
BR2 0.25
BR3 0.22
BR4 0.21
BR5 0.21
BR1 0.29
BR2 0.55
BR3 0.40
BR4 0.26
BR5 0.42
BR1 0.06
BR2 0.26
BR3 0.21
BR4 0.14
BR5 0.32
BR1 0.41
BR2 0.63
BR3 0.43
BR4 0.53
BR5 0.65
BR1 0.70
BR2 0.88
BR3 0.76
BR4 1.03
BR5 0.76
207.20
208.30
293.30
548.90
398.60
259.50
417.80
55.20
260.10
214.40
143.90
318.80
407.40
629.60
427.10
526.00
651.00
701.20
884.80
757.10
1030.50
760.50
246.30
359.90
257.90
604.50
328.10
496.40
481.60
436.60
768.60
435.20
719.50
400.70
150.60
246.30
223.20
800 amol 'heavy' peptide
Natural peptide per µg sample
[amol/µg]
81.02
Average amount per peptide
[amol]
SD
[amol]
212.74
502.66
183.49
340.12
130.80
309.20
368.80
309.75
321.05
145.48
56.32
CV
[%]
47
18
433.70
534.83
221.25
139.15
203.18
18.22
32
38
8
406.20
234.30
577.80
849.37
118.43
73.88
73.26
156.88
29
32
13
18
Ratio
'light'/
'heavy'
0.54
0.39
0.58
0.33
0.53
0.81
0.84
1.43
0.29
1.12
0.79
0.27
0.38
0.42
0.46
0.57
0.51
0.50
1.08
0.56
0.30
0.72
0.19
0.46
0.22
0.38
0.56
0.26
0.29
0.61
0.27
0.31
0.61
0.40
0.36
0.34
0.65
0.80
1.14
0.62
0.62
0.22
0.68
0.57
0.41
263.10
405.45
421.10
134.95
157.00
302.70
199.60
180.05
171.90
323.45
399.85
571.05
311.05
310.75
111.25
340.90
284.95
203.30
541.90
279.10
149.95
359.50
716.10
146.30
558.30
394.95
137.10
190.00
209.50
270.00
193.15
289.45
166.60
400 amol 'heavy' peptide
Natural peptide per
µg sample
[amol/µg]
Average amount per peptide
[amol]
SD
[amol]
96.60
232.45
109.20 202.96 73.32
CV
[%]
36
188.20
282.00
129.55
144.30
307.45
229.00
283.30
253.00
247.65
218.87 70.05 32
214.53 91.32 43
419.12
215.66
285.67
37.22
68
17
314.06 121.31 39
209.84
427.32
276.38
64.31
119.75
69.20
31
28
25
25

Supporting Information Figure 2. The kdp regulon.
This regulon consists of the constitutively expressed kdpDE operon encoding the histidine kinase KdpD, the response regulator KdpE, and the inducible kdpFABC operon encoding the high-affinity K + -uptake complex KdpFABC. Under K + limiting growth conditions a phosphorylated KdpE-dimer binds to a 23 bp sequence upstream of the canonical -35 and -10 regions of the kdpFABC promoter, triggering transcription in response to the stimulus. Upon kdpFABC transcription, a read-through of the RNA polymerase into the kdpDE operon increases kdpDE expression under inducing conditions.
P
2
( kdpFABC ), inducible
F A B kdpFABC mRNA
C
P
1
( kdpDE ), constitutive
E D kdpDE mRNA
“read-through“
26