Devices for use in MALDI mass spectrometry

Details Devices for use in MALDI mass spectrometry Devices for use in MALDI mass spectrometry

2000-08-24

2003-01-21

Warden, Jill (Department: 1743)

Chemical apparatus and process disinfecting, deodorizing, preser

Control element responsive to a sensed operating condition

C422S063000, C422S065000, C422S105000, C250S288000, C436S180000, C073S864250

Reexamination Certificate

active

06508986

ABSTRACT:

BACKGROUND OF THE INVENTION
A. Field of the Invention
The invention relates to a method and apparatus for preparation of samples that are to be subjected to mass spectrometry analysis. Specifically, the invention relates to methods for preparing samples for mass spectrometry analysis and to plate-like members used to position samples on a sample plate for subsequent mass spectrometry analysis.
B. Background Information
Mass spectrometry devices measure the molecular mass of a molecule by measuring the molecule's flight path through a set of magnetic and electric fields. Such devices are well known and are widely used in the field of bio-molecular research. In proteomics research, for instance, mass spectrometry is used to identify proteins.
Proteins are typically separated from one another by electrophoresis, such as the techniques described and claimed U.S. Pat. No. 5,993,627 to Anderson et. al. (the Anderson et. al. patent), which is incorporated herein by reference in its entirety. For instance, as set forth in the Anderson patent, a tissue sample is first subjected to a first dimension electrophoresis process where groups of proteins are separated linearly within a tubular gel filled column. The first dimension separation of proteins is then inserted along an edge of a flat planar gel slab and subjected to a second dimension of electrophoresis thereby generating a two dimensional pattern of spots formed by clusters of proteins that have moved to respective iso-electric focusing points. Thereafter, selected proteins are excised from the second dimension gel slab for further study. The selected excised spots are next prepared for analysis using, for instance, mass spectrometry.
An increasingly used technique for studying biological molecules includes the use of MALDI mass spectrometry apparatus (matrix-assisted laser desorption ionization apparatus) where the biological sample is embedded in a volatile matrix and is vaporized by being subjected to an intense laser emission. One such MALDI apparatus is a MALDI-TOF apparatus (TOF is time-of-flight spectrometry). In the field of proteomics, mass spectrometry, and in particular, MALDI-TOF techniques are used to determine the molecular weight of peptides produced by digestion of isolated proteins. One such MALDI-TOF apparatus is VOYAGER DE STR Biospectrometry Workstation manufactured and sold by APPLIED BIOSYSTEMS.
FIG. 1
depicts a generic MALDI-TOF apparatus that includes a frame
1
that supports the electronic and computer equipment necessary to control a laser
5
. The laser
5
is aimed at a fixed location in a positioning mechanism
10
. The positioning mechanism
10
includes means (not shown) for positioning a sample in the line of fire of the laser
5
. Typically in a MALDI-TOF apparatus, the laser is fixed in place and the sample is moved into position for analysis. The depicted MALDI-TOF apparatus includes a small removable sample plate
15
, shown in
FIG. 2
, that fits into the positioning mechanism
10
. Typically, the sample plate
15
is insertable into a slot
20
in the positioning mechanism
10
of the MALDI-TOF apparatus and is thereafter held in a specific orientation within the positioning mechanism
10
for sample analysis. The sample plate
15
typically holds a plurality of discrete samples on one surface thereof, with the samples being spaced apart from one another. The sample plate
15
includes guide members
15
a
, guide holes
15
b
and alignment pin
15
d
that are used by corresponding members (not shown) within the positioning mechanism
10
for positioning and moving the sample plate
15
with respect to the line of fire of the laser
5
.
The samples are typically loaded onto the sample plate
15
by a separate device or robotic apparatus, such as the X-Y robotic apparatus depicted in FIG.
3
. Such X-Y robotic apparatuses are typically manufactured and sold with each specific mass spectrometry apparatus. The X-Y robotic apparatus depicted in
FIG. 3
includes a recess that retains the sample plate
15
in position for sample loading. The X-Y robotic apparatus includes a first arm
30
that moves back and forth along an X axis and a second arm
40
that moves along a Y axis defined along the length of the first arm. The second arm
40
supports a pipette tip
45
that is used to spot samples on the sample plate
15
. Specifically, the pipette tip
45
is moved by the first and second arms
30
and
40
to a position above a 96-well plate
50
(or other similar sample holder) or microcentrifuge tubes and the pipette tip
45
picks up a sample from the 96-well plate
50
or microcentrifuge tubes. The pipette tip
45
is then moved to a location above the sample plate
15
and the sample is spotted on the specified location of the sample plate.
Typically, an array of samples are spotted on the sample plate
15
at predetermined locations. After the array of samples are loaded onto the sample plate
15
, the sample plate
15
is inserted into the slot
20
of the MALDI apparatus. Using an imaging system
25
focused on the sample plate
15
within the MALDI apparatus, in combination with the positioning mechanism
10
, the laser beam from the laser
5
can be aimed, one by one, at the sample(s) on the sample plate
15
.
The mass spectrometry apparatus typically takes several hours to analyze an array of samples on the sample plate
15
. Therefore, in order to minimize human involvement, automation of the process is critical. The locations of the samples are typically pre-programmed into the computer that controls the MALDI-TOF apparatus so that during the analysis of the samples, the positioning mechanism
10
automatically repositions the sample plate
15
into the line of fire of the laser
5
. Therefore, if any of the samples on the sample plate
15
are not properly positioned, the laser
5
is not likely to hit each of the samples. For instance, on the sample plate
15
depicted in
FIG. 2
, a 10×10 array of samples is positioned on the upper surface at spaced apart intervals. The positioning mechanism
10
moved into a target position with respect to centers of the desired location of each spot. The desired location of each spot assumes that center of each of the spots in the 10×10 array is constant.
Unfortunately, there are several shortcomings associated with the above described X-Y robotic apparatus (FIG.
3
). Although the positioning mechanism
10
within the MALDI apparatus has positional accuracy with respect to movement of the sample plate
15
, the X-Y robotic apparatus typically sold with a MALDI apparatus is not as precise with respect to accurate spotting or depositing of samples on the sample plate
15
. Specifically, the spots in a 10×10 array of samples do not wind up being centered on the desired center targeted by the positioning mechanism
10
. The array of 10×10 samples may have some samples that are accurately centered, and other samples that are off center by as much as half the width of the sample.
Part of the problem with such X-Y robotic apparatuses relates to the replaceable pipette tips
45
used to retrieve a sample and deposit the sample onto the sample plate
15
. The pipette tips are not perfectly uniform in size and shape. Further, the tips are deformable and hence accurate positioning of samples is not possible. As well, the X-Y robotic apparatus may not have movement and location capabilities as precise as the movement and location capabilities of the positioning mechanism
10
of the MALDI apparatus.
Another problem relates to the pre-programmed settings for locating samples on sample plates
15
of such X-Y robotic apparatuses. Specifically, X-Y robotic apparatuses are not programmed to maximize the use of the space of the surface of the sample plate
15
. For instance, each pair of adjacent samples in the 10×10 array of samples mentioned above is typically spaced apart by a distance greater than the diameter of the sample. There is a substantial amount of empty space on the surface of the sample plate
15
even with a 10&t

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