Quantitative comparison of sensitivity and saturation for

Quantitative comparison of sensitivity and
saturation for MALDI-TOF detectors when
measuring complex and high mass samples.
Ryan J. Wenzel1, Susanne Kern2, Alexis Nazabal1 and Renato Zenobi2
1CovalX
AG Zürich,
AG,
Zürich Switzerland
of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology,
ETH Zürich, Switzerland
2Department
Introduction
Recently, there has been interested in measuring intact protein complexes by mass spectrometry.
Often, the mass of protein complex is several hundred kDa and the samples mixture quiet complex.
Most commercial MALDI-TOF instruments use MCP detectors which are not ideal for high mass ions
g
masses a
because of sensitivityy and saturation issues. In order to reliablyy measure ions at these higher
more sensitive detector is beneficial. Also, when measuring complexes mixtures, it is necessary to use
a detector which will not saturate from ion signals coming from multiple peaks over a broad mass range.
To address these issues, various ion detectors were evaluated using identical conditions and the effects
of sensitivity and saturation were quantitatively evaluated.
IgM+3
IgM+2
IgM+
ICD
Sensitivity at a MegaDalton
STJ
Detector Descriptions
A Bruker Reflex IV was retrofitted with a CovalX
HM1 high mass detection system. The HM1
detector can be moved in-line of the ion flight path
directly in front of the standard linear
MicroChannel Plate (MCP) detector. The HM1
detector is composed of an Ion Conversion
Detector (ICD) and all the necessary electronics
to operate. The HM1 allows for easy comparison
of ICD with MCP in the same instrument under
identical conditions within seconds. Also used
during this comparison was the macromizerTM
instrument, which contains a Superconducting
Tunnel Junction (STJ) cryodetector. Both the ICD
and STJ technologies were designed specifically
for the sensitive detection of high mass ions
because they do not rely on the impacting ions
velocity.
velocity
m/z (kDa)
Spectra of 1 MegaDalton (Immunoglobulin M) detected on
the STJ (A) and ICD (B) detectors. No high mass signal
was detected when
measuring with the standard
MCP.
macromizer STJ detector
Limits of Detection (LOD in uM)
CovalX HM1 High Retrofit
Protein
Insulin
Cyto C
FLAG
HSA
AntiFLAG
IgM
m/z (kDa)
5
12
50
66
150
1000
MCP
1.25
1.25
1.25
09
0.9
1.6
n.a.
ICD
0.3
1.25
0.6
0 45
0.45
0.45
0.16
STJ
0.3
1.25
0.3
0 45
0.45
0.45
0.16
A two times dilution series was measured (1µL each). It is important to note that these are not universal LOD.
These were measured under non-optimized conditions (i.e. No Delayed Extraction, etc) to keep the measurements
as similar as possible, without optimizing the experiment for each sample. The ICD and STJ typically give
comparable results, with the MCP showing often 2 to 3 times higher LOD.
TURN OVER
Insulin / BSA Saturation Test
Insulin and Bovine Serum Albumin (BSA) were mixed at high
concentrations (19µM BSA and 18 or 9µM Insulin) and the relative
intensity of BSA was monitored as the laser power was changed
(Figure below). At higher laser powers relatively more BSA ions are
created, as detected by the ICD. The MCP shows virtually no BSA
signal at any laser power because the MCP is saturated from the
earlier arriving Insulin ions; with no Insulin the MCP easily detects
the BSA.
Spectra (shown on right) demonstrating this saturation can be seen
in to the right where the MCP shows no peaks above the insulin
signals because of detector saturation.
100
BSA??
Measurements taken on same spot in same instrument with
exact same instrumental values within seconds of each other.
90
BSA Relative Intensity (%)
MCP
Insulin
80
HM1
Insulin
70
60
50
Insulin
Adducts
BSA+1
40
BSA+2
30
2BSA+
20
10
3BSA+
0
60
65
Blue = BSA measured using HM1
Red = BSA measured using MCP
70
75
80
85
90
Laser Power (%)
Spectra from Multicomponent Mixture
STJ
AntiFLAG(+2)
AntiFLAG
ICD
FLAG
HSA
A
MCP
Insulin
Lysozyme
Both the ICD and STJ show easily identifiable signals corresponding to each of the five proteins in the mixture.
The MCP is only able to detect the two lower mass proteins. Many mixtures of different relative concentrations
were measured, always with the same results. It is believed the large difference in detection is because of
t ti off th
h
l as th
bl tto d
t t each
h off th
di id l proteins
t i att th
l
saturation
the MCP channels
the MCP was able
detect
these iindividual
these llevels
when measured separately. It is important to note that all these samples were measured from the exact same
solutions within minutes of one another, and the MCP and ICD were taken on the exact same instrument with
absolutely no changes to operating conditions.
Conclusions
DYNAMIC RANGE: Samples extending over nearly three orders of magnitude in mass range
(5kDa to 1MDa) were easily detected.
SENSITIVITY: Both the ICD and STJ detectors show similar LOD with detection up to 1MDa.
The MCP is usually 2 to 3 times less sensitive for pure samples under identical conditions.
p
component
p
mixtures are measured a strong
g sensitivityy
SATURATION: When multiple
decrease is seen for the MCP. It is believed this is due to substantial detector saturation.
PRACTICAL ASPECTS: The STJ and ICD provide comparable data. The STJ was an
expensive instrument which is no longer being produced. The ICD is an affordable alternative
which can easily be retrofit to most major MALDI TOF instruments. MCP is good for simple
low mass analysis, but not sensitive for high mass and saturates highly with complex mixtures.