CLINICAL CHEMISTRY
Original Article
Quality Assurance and Reproducibility
of High-Resolution Two-Dimensional
Electrophoresis and Silver Staining
in Polyacrylamide Gels
HAROLD H. HARRISON, M.D., P H . D , 1 3 KATHY L. MILLER, MT(ASCP),1
CHARLENE DICKINSON, M.A., 2 AND
JUDITH A. DAUFELDT, M.S., MT(ASCP), 34
Qualitative data from high-resolution two-dimensional electrophoresis (2DE) have become clinically useful in immunoglobulin
clonality analysis, in resolution of ambiguities in immunofixation
typing of paraproteins, and in genetic typing of serum proteins.
Since 1986, the authors have been evaluating the College of
American Pathologists Reference Preparation for Serum Proteins
(RPSP) as a quality control material for 2DE because (1) it is
prepared exclusively from pooled human sera, (2) the pool yields
a reference pattern of mixed heterozygosity for genetic markers,
and (3) RPSP is widely available as a lyophilized preparation
that currently serves in the authors' laboratory as a qualitative
quality control preparation and that may become a quantitative
quality control material or external quality assessment material
for 2DE. Using the ISO-DALT 2DE system and silver-staining,
the peptide patterns were examined in 11 lots of RPSP and compared with fresh serum and with each other. Consistent differences in the 2DE pattern between RPSP and fresh serum included
the presence of freeze-thaw peptides, the presence of degradation
spots of apolipoprotein A-I, and the diminution of apolipoprotein
spot intensities in RPSP. All lots of RPSP yielded clear identification of the eight serum proteins used for quality control
calculations. Run-to-run coefficients of variation for a single lot
of RPSP for four parameters of 2DE spot location and gradient
reproducibility were comparable with band location reproducibility for the one-dimensional procedures of serum protein electrophoresis and lactate dehydrogenase isoenzyme electrophoresis. It is concluded that the reproducibility, that is, imprecision,
of 2DE is the same as one-dimensional clinical electrophoresis
techniques and that either RPSP or pooled fresh serum can serve
as a satisfactory internal quality control material. (Key words:
Two-dimensional electrophoresis; ISO-DALT; Silver-staining;
Quality control; Quality assurance; RPSP; Serum protein electrophoresis) Am J Clin Pathol 1992;97:97-105
in a single polyacrylamide gel, depending on the protein
detection technique. 1-3 The high resolving power of 2DE
results from the sequential combination of two electrophoretic techniques that are inherently high resolution
From the Departments of'Pathology and "''Anthropology, Universitymethods: (1) isoelectric focusing, which separates proteins
ofChicago Pritzker School ofMedicine. Chicago, the1Molecular Anatomy
on the basis of intrinsic charge, and (2) sodium dodecyl
Program. Division of Biological and Medical Research, Argonne National
sulfate polyacrylamide gel electrophoresis, which separates
Laboratory, Argonne, and1Abbott Laboratories. North Chicago, Illinois.
peptides by molecular mass. When coupled with a senReceived February 14, 1991; received revised manuscript and accepted
sitive
silver stain, this combination allows the simultafor publication June 3, 1991.
neous
qualitative and quantitative analysis of several
Supported in part by the Abbott Laboratories (J. Daufeldt), the American Cancer Society, Illinois Division (K. Miller), the L.S.B. Leakey and
hundred proteins in microvolume human body fluid and
Wennergren-Gren Foundations (C. Dickinson), the Sprague Institute
tissue samples.
(H. Harrison), University of Chicago Medlabs TRSP Fund (H. Harrison
and K. Miller), and the United States Department of Energy (equipment
Recently, distinct clinical indications for performing
loan to H. Harrison), contract W-31-109-ENG-38, Argonne National
2DE as a diagnostic procedure have emerged. These inLaboratory.
clude (1) analysis of serum and urine immunoglobulin
Preliminary data presented in part at the American Society of Clinical
patterns for adjunctive clonality analysis and resolution
Pathologists/College of American Pathologists 1990 Fall meeting in Dallas, Texas.
of ambiguities encountered in immunologic typing of
Address reprint requests to Dr. Harrison: Department of Pathology,
paraproteins in the context of B-cell neoplasia diagnoBox 146, The University of Chicago Pritzker School of Medicine, 5841
sis 4-6 ; (2) detection of low-expressor Z phenotypes in proSouth Maryland Avenue, Chicago, Illinois 60637.
High-resolution two-dimensional electrophoresis (2DE)
allows the analysis of as many as several thousand proteins
97
98
CLINICAL CHEMISTRY
Article
tease inhibitor (alpha-1-antitrypsin) genetic typing7; (3)
analysis of serum protein genetic types in population association, forensic, and anthropologic correlation studies8"12; (4) analysis of lymphocyte proteins in forensic
studies' 3 ; and (5) analysis of cerebrospinal fluid proteins
in patients with Jakob-Creutzfeldt and other neurologic
diseases.1415
The ISO-DALT 2DE system (Large Scale Biology, Inc.,
Rockville, MD) used in our laboratory allows the casting
and running of 20 gels simultaneously, a feature that yields
improved within-run and run-to-run reproducibility. As
with other clinical laboratory methods, the medical use
of 2DE requires appropriate protocols for quality control
(QC) and quality assurance. To this end, we developed
methods for archival image recording of silver-stained
2DE gels,16 generated protocol recommendations that result in improved reproducibility, and initially suggested
the use of the College of American Pathologists Reference
Preparation for Serum Proteins (RPSP) as a QC material
for the procedure.17 These early studies with RPSP were
performed in the research laboratory using clinical laboratory QC procedures.
We report here a detailed evaluation of RPSP as a QC
material for ISO-DALT 2DE in a start-up hospital laboratory and the results of precision studies for the qualitative use of 2DE as a diagnostic procedure. Statistical
indicators for 2DE run acceptance were based on spot
position in both dimensions and on slope and intercept
parameters obtained for a Logit-Log regression equation
for position versus mass in the DALT dimension. We also
compared the qualitative reproducibility of 2DE spot locations with one-dimensional serum protein electrophoresis and lactate dehydrogenase isoenzyme procedure
band locations to demonstrate that 2DE patterns, although visually complex, are as reproducible as any other
clinical laboratory electrophoresis procedure.
Sample Preparation
Sera or reconstituted RPSP samples were denatured
and solubilized in four volumes of sodium dodecyl sulfate
mix, which contained 2% sodium dodecyl sulfate, 10%
glycerol, and 1% dithiothreitol in 50 mmol/L cyclohexylaminoethane sulfonate (pH 9.5).' 9 All diluted samples
were heated for 5 minutes (±15 seconds) at 95 °C. Six
microliters of the denatured serum samples (1.2 /*L serum
equivalent) was applied to the isoelectric focusing (ISO)
gels, and the remaining denatured aliquots were stored at
- 7 0 °C.
Two-Dimensional Polyacrylamide Gel Electrophoresis
MATERIALS AND METHODS
High-resolution 2DE of proteins' was performed with
the 180 X 180 X 1.5 mm ISO-DALT system19 for casting
and running 20 2DE gels simultaneously, with minor
procedural modifications designed to improve reproducibility. 1720 All reagents were "electrophoresis" grade. Isoelectric focusing (first dimension) gels included an ampholyte mixture containing, per liter, 8 g of Servalyt, pH
3-10 (Serva Feinbiochemicals, Heidelberg, FRG); 8 g of
Pharmalyte, pH 3-10; and 4 g of Ampholine, pH 5-7
(LKB Pharmacia Biotechnology Inc., Piscataway, NJ).
The conditions of isoelectric focusing were 14,000 V • h
± 1%, with a range of 700 to 900 V. The ISO gels were
equilibrated with SDS buffer (pH 6.8) at room temperature for 10 minutes ± 15 seconds and stored at - 7 0 °C
until loaded onto DALT gels. Isoelectric focusing gels were
aligned with the acid end at the top left corner of each
DALT gel. Electrophoresis of proteins through the linear
90 g/L to 180 g/L polyacrylamide gradient slab gel (second
dimension, DALT, sodium dodecyl sulfate polyacrylamide gel electrophoresis) in sodium dodecyl sulfate
TRIS-glycine buffer (pH 8.6) was performed at 150 V for
17 hours. To ensure the quality of casting and later to
verify uniformity of electric current movement, a trace
of bromphenol blue was added to the denser acrylamide
solution during the casting.
Reference Preparation for Serum Proteins. Vials of
RPSP, a certified reference material that has been partially
delipidated and lyophilized, were reconstituted with glassdistilled water as specified. The detailed description of the
certification process for this material as a quantitative reference preparation for specific proteins has been reported.18 Lot-to-lot comparisons of peptide patterns for
RPSP were performed within runs.
Serum. Blood samples from volunteers and patients
were collected in Vacutainers (Becton Dickinson, Inc.,
Patterson, NJ), centrifuged, and the supernatant sera
stored at - 7 0 °C until time of analysis by 2DE.
Proteins in the DALT gels were stained by a modification of an ammoniacal silver-stain method. 21 The modification consisted of two fixation steps: 0.1 % formaldehyde in 50% ethanol for 1 hour, followed by 2.5% sulfosalicylic acid, 5% acetic acid, and 20% ethanol for 4 hours.
Subsequent steps were unaltered. Archival XRD (Kodak
X-Ray duplicating film, Eastman Kodak, Rochester, NY)
images, from which black-and-white representations are
derived, were made for each silver-stained gel.16 Computer-generated schematic images were made on Macintosh Plus, SE, SE30, or II microcomputers (Apple Computers, Inc., Cupertino, CA) using the MacGEL image
analysis program and related image manipulation software
developed in our laboratory (unpublished, copies available
Specimens
A.J.CP. -J; luarv 1992
HARRISON ET AL.
Quality and Reproducibility of High-Resolution 2DE and Silver Staining
on request). All gel images and vignettes were displayed
with increasing isoelectric point from the left to the right20
and increasing molecular size (MW) from the bottom to
the top (Figs. 1 and 2).
99
MPL7I4
Quality Control Measurements and Calculations
Quality control measurements and calculations were
performed by visual determination of spot location against
a transillumination grid that had 2-cm major and 1 -mm
minor interval markings and by manual recording of positions to the nearest 0.5 mm for the following proteins:
«i-acid glycoprotein, a r antitrypsin (monomer and dinner), a 2 -HS glycoprotein, apolipoprotein A-I, complement factor 4 (C4) 7 chain, Gc globulin, haptoglobin a2
and (8 chains, immunoglobulin (Ig) gamma heavy chains,
Ig K light chains, Ig J chains, prealbumin (transthyretin),
and transferrin.20 The same transillumination approach
was used to measure band locations in one-dimensional
serum protein electrophoresis (albumin band) and lactate
dehydrogenase isoenzyme electrophoresis (LD5 band) in
agarose performed according to the manufacturer's specification (Paragon, Beckman Instruments, Inc., Brea, CA).
The transillumination measurements were made by overlaying a 5x lens and the gels and the reference transparency
grid on a fluorescent light box. For measurements of the
2DE spot locations, the origin was the upper left-hand
corner (topmost, acid end) with gels in the orientation
shown in Figure 1. Thus, for the 2DE gels, increasing x
and y values reflected increasing isoelectric point and decreasing mass, respectively. In the one-dimensional gels,
measurements were made to the band centers in control
sample lanes. For the serum protein electrophoresis gel
albumin measurements, the origin was the sample application point and for the lactate dehyrdogenase isoenzyme
gel measurements the parameter used was the distance
between the LD1 and LD5 isoenzyme bands.
The numeric parameters used for QC calculations for
2DE were the absolute positions in the ISO (x) and DALT
(y) dimensions of apolipoprotein A-I (spot O), and the
slope and intercept of the least squares regression of Log
MW versus Logit Rm curve for the DALT dimension positions of the proteins noted above. The empirical linearization Logit Rm parameter was calculated as the Log [Rm/
(1-Rm)], where Rm (relative electrophoretic migration) was
the absolute DALT distance migrated (y) divided by the
total length of the gel. The Log MWSDs was calculated
from the reference mass of each peptide.17 This approach
was used to provide a linear plot for the otherwise sigmoidshaped curve of Rm versus MWSDS or Log MWSDS. Qualitative acceptance criteria for each 2DE gel were based
on the clear visualization of all isoforms of the a r a n t i -
FIGS. 1A and B. Silver-stained 2DE patterns of serum and RPSP. (A)
Serum admixed from ten individuals; (B) RPSP, lot no. 9628T001A.
Arrows indicate major freeze-thaw (upper arrow) and apolipoprotein AI (lower arrow) peptides that occur in RPSP and not in serum.
trypsin, Gc globulin, transferrin, and haptoglobin spot
groups.
Statistics and Graphics
Means (x), standard deviations (SD), and coefficients
of variation (CV) were calculated with standard Gaussian
Vol. 97 . No. I
100
CLINICAL CHEMISTRY
Article
parametric statistical formulas using the Statview 512+
(Abacus Concepts, Inc., Berkeley, CA) statistics program.
Levey-Jennings charts for QC plotting were generated with
the Cricket Graph program (Cricket Software, Inc., Philadelphia, PA), and plots of 95% confidence zones (not
shown) for each QC peptide were made with the MacDraw
program (Apple Computers).
RESULTS
Two-Dimensional Electrophoresis
Patterns
The silver-stained 2DE patterns of pooled fresh serum
(frame A) and RPSP (frame B) in the 18 X 18 cm ISODALT system are shown in Figure 1. Arrows in frame B
indicate the two main areas of difference between the patterns. The upper arrow indicates a group of spots referred
to as "freeze-thaw" peptides17,22 that appear in the 2DE
pattern at a slightly lower mass and isoelectric point than
the acidic haptoglobin-/? isoforms after frozen serum and
plasma samples have been thawed. These peptides occurred in all lyophilized preparations or after a single
freeze-thaw cycle of fresh serum, regardless of whether
denaturation preceded or followed freezing. The source
identity of these peptides has not yet been established, yet
their occurrence is ubiquitous. The lower arrow points to
a series of peptides related to the apolipoprotein A-I group
that occurs in RPSP but not in serum. These apo A-I
peptides are presumably generated during the manufacturing process, which includes steps for delipidation and
lyophilization, because they do not occur in serum specimens that have been repeatedly frozen and thawed in
our laboratory.
Figure 2 shows a schematic representation of the silverstained RPSP map with numbered oval boxes added to
highlight zones in which lot-to-lot peptide pattern differences occurred in at least one lot when 11 lots were compared in a single run. These variable peptide zones are
numbered but not elaborated in further detail because the
differences were generally subtle and did not interfere with
either identification of the proteins used for the QC measurements (indicated with set-off square boxes) or identification of major non-apolipoprotein serum proteins for
which RPSP may serve as a quantitative standard or control. Areas 5 and 9 are the areas in which the freeze-thaw
and apolipoprotein A-I degradation peptides occurred
(Fig. 1). Name labels indicate polypeptide identities that
have been previously well established.'7,22"24 Groups of
peptides connected by solid lines represent charge and/
or mass isoforms of proteins that are known to arise from
the same gene.
A.J.C.P. - J ;
Reproducibility Calculations for 2DE
Within-run and run-to-run precision data are shown
in Table 1. The within-run data were gathered for two
separate serum protein genetic typing experiments performed 1 month apart. The run-to-run data show the results of one RPSP gel from each of 49 separate experiments
performed during a 48-month period with the same lot
of RPSP. As is usual, the within-run CVs generally are
smaller than their between-run counterparts. Furthermore, yearly subsets of the between-run data have lower
CVs than the overall group shown. These 49 runs are the
same used in Table 2, in comparison with four-parameter
QC reproducibility data for 20 runs in which serum specimens were measured (Table 1).
Positional Precision of 2DE Spots Compared with
One-Dimensional Electrophoresis Bands
Table 3 shows between-run positional precision calculations for both 2DE spot locations and the band locations for the one-dimensional procedures of serum protein electrophoresis and lactate dehydrogenase isoenzyme
fractionation. Because of the length of the 2DE procedure
(4 days), the 2DE data are week-to-week, whereas the onedimensional data are day-to-day measurements taken
during a period of 2 months (Table 3).
RPSP Versus Serum as the Control Material for 2DE
Figure 3 shows examples of Levey-Jennings plots for
the four previously mentioned QC parameters. The data
used to generate these charts were from combined RPSP
and serum data (one QC gel per run) from August 1986
to July 1989. By way of further illustration of the reproducibility of the 2DE technique, the gels shown in Figure
1 were run more than 1 year apart. The gel on the left
(RPSP, frame A) was run on February 2, 1988 and the
gel on the right (serum, frame B) was run on April 24,
1989.
Table 2 shows the between-run precision results for 69
separate ISO-DALT 2DE runs conducted during a period
of 4 years. In 49 runs, RPSP was run in at least one lane,
whereas in 20 other runs, a serum sample was used as the
control (Table 2).
DISCUSSION
Rationale and Acceptability of Using RPSP
as QC Material
We selected RPSP as a QC material and potential external quality assurance material for 2DE for four reasons.
First, it is a College of American Pathologists- and United
States Centers for Disease Control-certified standard for
\- 1992
HARRISON ET AL.
Quality and Reproducibility of High-Resolution 2DE and Silver Staining
M W SDS
••••
(KD)
101
« 2 Microglobulin
=TT*£
106
o^ATdimerl
„...••
Ceruloplasmin
!
Hemopexin
74.2
53.2
a-1 Antichymotrypsin
Glycoprojein|»a&0%^
j
E
D
»s—o-o-o-o—C3 0
x V
Ig y I heavy chain region
^tHiJjP''" chains
Gc globulin
* • «,
Hp 3 chains.
40.3
G
I Transferrin!
• • •
•
«1 Acid
Glycoprotein
«
o
•
o O
D"'
©
6
o O
(0)
peptides 7 y ^ " ? \ < $ ^
o O
O
2
ApoE
fi-C4ff
I Ig K land X light chain region
8
26.0
24.3
*©'
IQP
o
9/ .swr 1 '
|lg J chains]
cu§
o
. o
o
Haptoglobin a 2 chains]
18.5
.-O—CD-
10
[prealbumin I
14.6
Hemoglobin p chain
'
Apo C lipoproteins
1
~
Haptoglobin a , chains
Apo All
CK Charge Stds:
-35
-30
25
•
1
-20
•
•
1
-10
-15
•.
'
•
-5
_i_
•,
8
*
*
•
9
~pl
FIG. 2. Schematic representation of RPSP 2DE map. Major serum proteins are labeled. Numbered oval boxes highlight regions that exhibited lotto-lot differences. Offset square boxes highlight peptides used for quality control measurements. F-T, = freeze thaw peptides.
12 serum proteins: albumin, a t -acid glycoprotein, a,-antitrypsin, a2-macroglobulin, C3, C4, ceruloplasmin, haptoglobin, IgG, IgA, IgM, and transferrin.21 Second, it is
prepared exclusively from several hundred pooled human
sera per lot and contains no additives or "doping" from
nonhuman sources that might introduce xenogenic peptides into the 2DE patterns. Third, the serum pool yields
a reference pattern of mixed heterozygosity for 2DE analysis of serum protein genetic markers. Fourth, RPSP is
commercially available from the College of American Pa-
thologists as a lyophilized preparation that eventually may
serve as a quantitative QC material.
The data presented in Tables 1 and 2 indicate that
equivalent precision results are obtained with either RPSP
or serum as a qualitative internal QC specimen. With
regard to use as an external quality assurance material,
RPSP has the same degree of reproducibility of spot locations and densities as does serum and also has the advantage of being a certified traceable standard. Indeed,
RPSP recently has been used as a control preparation by
102
CLINICAL CHEMISTRY
Original Article
TABLE 1. REPRODUCIBILITY DATA FOR 2DE
Slope
(DALT)
Experiment
Intercept
(DALT)
Apo AI
(ISO)
TABLE 3. BETWEEN-RUN POSITIONAL REPRODUCIBILITY
DATA FOR ONE-DIMENSIONAL AND TWO-DIMENSIONAL
ELECTROPHORESIS PROCEDURES
Runs
Within-run (Expt. 48, n = 19)
Mean
SD*
CV, %
Within-run (Expt. 50, n = 17)
Mean
SD
CV, %
Run-to-run (n = 49)
Mean
SD
CV, %
-0.568
0.014
2.42
4.458
0.015
0.337
3.467
0.156
4.503
-0.594
0.010
1.76
4.448
0.011
0.256
3.266
0.199
6.105
-0.581
0.021
3.670
4.459
0.024
0.528
3.724
0.213
5.730
2DE = twcKiimensional electrophoresis; SD = standard deviation: CV = coefficient of variation.
another group of investigators who used Coomassie staining and a disk-operating system-based microcomputer
system for quantification of proteins in 2DE gels.25 However, quantitative measurements of proteins in 2DE patterns of serum and other body fluids are still problematic.
Two-dimensional electrophoresis patterns of body fluid
proteins, although currently useful in a qualitative mode
as mentioned, are difficult to quantify because of the
complexity of the distribution of isoforms and microheterogeneity introduced by presecretory, post-translational
sialylation and other modifications. The high resolving
power of the technique separates isoforms of many serum
proteins that differ by as little as a single electrostatic unit,
such as a single neuraminic acid residue or single amino
acid substitution.''20'23'26 Thus, summation of the integrated pixel densities of a group of spots is required to
achieve quantitative analysis for many proteins that occur
as a single agarose electrophoresis band. Furthermore, the
silver staining procedure, although linear for most proteins, yields varying density versus quantity responses on
a between-protein basis due to differing numbers of nucleation sites for silver grain deposition.21 These factors,
although largely a surmountable engineering problem,
make quantitation of 2DE gels impractical in routine
clinical use until better and more inexpensive image analysis and quantitative correlation systems become available.25 When such systems do become available, RPSP
Technique
Protein Measured
(«)
CV (%)
SPE
LDH isoenzymes
ISO-DALT 2DE
Albumin
LD5
Apolipoprotein A-I
17
18
41
7.41
5.28
4.86 (ISO)
3.87 (DALT)
CV = coefficient of variation; LDH = lactate dehydrogenase; LD5 = LDH isoenzyme fraction
5; SPE = serum protein electrophoresis; 2DE = high-resolution, two-dimensional electrophoresis.
or a similar certified standard would be a likely choice
for quantitative external quality assurance.
RPSP Versus Serum as a QC Material
There is virtually no difference in the results for RPSP
versus serum in terms of between-run reproducibility assessment (Table 2). Between-lot differences in character
or number of isoforms for the major serum proteins used
in QC measurements were not observed. The main differences between lots of RPSP involved small groups of
peptides that were of no consequence to overall analysis
of the gel. Apolipoprotein A-I yielded a consistent pattern
of partial proteolysis from lot to lot, yet the apo A-I (spot
0) was easily identifiable. The only additional factor to be
considered in the choice of RPSP versus serum as a QC
material is whether the 2DE analysis is being used for
serum protein genetic typing. If so, sera that are to be
mixed for an "in-house" QC pool should contain all of
the major types of the proteins being phenotyped, which
may include Gc globulin, haptoglobin, a {-antitrypsin, a2HS glycoprotein, apolipoproteins A-IV and E, SPPM-158,
SER-1, or transferrin. 29 '" 12 - 22 - 23
Overall Reproducibility of 2DE as Revealed by the
Use of Serum or RPSP
It is important to remember that the precision data
shown here for 2DE pattern reproducibility were obtained
by manual measurements of spot location relative to an
absolute reference point of the upper left-hand corner of
TABLE 2. REPRODUCIBILITY DATA FOR RPSP VERSUS SERUM
Slope
Intercept
A-I (DALT)
A-I (ISO)
Specimen
Runs
(n)
X
CV
X
CV
X
CV
X
CV
RPSP
Serum
49
20
-0.581
-0.590
3.67%
2.89%
4.459
4.453
0.53%
0.46%
3.724
3.864
5.73%
7.63%
5.347
5.310
4.06%
3.59%
RPSP = Reference preparation for serum proteins; X = mean; CV = coefficient of variation.
A.J.C.P.'January 1992
HARRISON ET AL.
Quality and Reproducibility of High-Resolution 2DE and Silver Staining
Apolipoprotein A1 Position
103
Logit Rm Plot Parameters
4.56'
4.4'
+3SD
4.2'
+2SD
+3SD
4.52'
+2SD
4.0'
4.48-
3.8
3.6
4.44-2SD
3.4
-2SD
3.2
4.40-
-3SD
-3SD
SD=0.181;CV=4.86%
3.0
10
15
-1
20
25
Experiment
1
30
1
35
i
40
SD= 0.023; CV= 0.513%
4.3610
45
15
20
25
Experiment
30
35
40
45
-0.48
6.20
+3SD
+2SD
5.80-
+3SD
-0.52
+2SD
-0.56
5.40-
-0.60
5.00-
-2SD
-0.68
4.60
15
20
25
Experiment
30
35
40
-3SD
-3SD
SD= 0.209; CV= 3.87%
10
-2SD
-0.64
10
45
15
SD= 0.023; CV= 3.95%
—i
1
1
1
20
25
30
35
40
45
Experiment
FIG. 3. Example Levey-Jennings charts for 2DE reproducibility parameters. (A) Apo A-I ISO position; (B) Apo A-I DALT position; (C) Log MWLogit Rm least squares intercept; and (D) Log MW-Logit Rm least squares slope.
the 18 X 18 cm gel. Thus, the data encompass relatively
long-range gel errors of physical stretching, albumin zone
breakage of the ISO dimension and distortion of isoelectric
point domains, or molecular sieving interference in areas
of high protein concentration. The proteins used for the
QC measurements were specifically selected to represent
all areas of the gel in terms of net acidic and basic charges
in the first dimension and large mass and small mass mobilities in the second dimension. The actual gel distance
traversed varied substantially from protein to protein, yet
all gave comparable 95% confidence zone results (not
shown). For apolipoprotein A-I, there was a physical distance of movement into the gel of between 10.5 and 11
cm in both dimensions. In the case of prealbumin (transthyretin), the distance traveled was 14 cm in the DALT
dimension. In contrast, the one-dimensional agarose procedures, which have comparable positional reproducibility, require only one third to one half as much travel in
a single direction. Yet despite the much larger physical
distances, with attendant magnified opportunity for in-
terference from polyacrylamide gel matrix, reagent, sample, or electric field inhomogeneities, the 2DE procedure,
which requires 3 to 4 days and several manual intervention
steps to complete, is as reproducible as the one-dimensional methods.
It is important to reiterate that the calculations we performed were based on manual measurements of protein
location in silver-stained 2DE gels and not on photographs, autoradiographs, or computer-generated images
or data. Indeed, when image analysis and pattern-matching algorithms are used, the calculated degree of imprecision for 2DE patterns can be reduced by an order of
magnitude.25-27 Nevertheless, good reproducibility for
routine qualitative 2DE can be achieved without using
sophisticated image analyzers or computers. The electrophoresis equipment used here is commercially available,
relatively inexpensive, and requires only careful work by
the technologist to achieve reliably interpretable patterns.
The major advantage of the computer systems is not in
analysis of individual gels but rather in the comparison
Vol. 97 • No. I
104
CLINICAL CHEMISTRY
Original Article
of large numbers of spotlists and databases.28 This degree
of sophistication is not necessary for interpretation of a
limited number of known proteins in a single gel per patient.
An outline of the 2DE procedure includes the following
major experimental steps: (1) sample preparation in a
buffer that produces completely denatured and reduced
peptides, (2) isoelectric focusing in the presence of a pH
gradient created by reagent ampholytes in a tube gel, (3)
sodium dodecyl sulfate polyacrylamide gel electrophoresis
in a polyacrylamide gradient slab gel of restrictive porosity,
(4) detection of the proteins with Coomassie or silver
staining, and (5) analysis of the resulting 2DE gel pattern
by comparison with reference specimens or a reference
database. We found that the key factors that relate to reliability of the method are sample preparation, ampholyte
selection, and the silver-stain procedure. First, all samples
should be prepared and applied to the ISO gels in a standardized manner. Second, ampholytes from different
manufacturers have different chemical structures and different numbers of components.29 Thus, despite these crucial reagents having a similar nominal isoelectric point
range specification, different first-dimension results are
obtained with different batches of ampholytes—between
lots and between manufacturers. Therefore, once a suitable batch or blend is found, we suggest purchasing enough
ampholyte to run a large number of gels. Third, silver
staining, which is 20 to 50 times more sensitive than Coomassie staining, may vary from run to run in analytic
sensitivity. However, this is only problematic for typing
of low concentration or weakly staining proteins. The
causes of silver-stain variation appear to be due to environmental factors of temperature, reagent water, and
ethanol quality. These variations are not a problem if care
is taken to apply sample volumes that yield clear staining
of all proteins of interest.
Generally, problems of gel reliability relate to a localized
artifact, such as an air bubble, or enhanced or diminished
staining in a small area of individual gels. Such localized
anomalies usually are not considered substantive enough
to reject interpretation of other areas of the gel. Qualitative
visual analysis of the protein molecular pathology patterns
represented in the 2DE gels is analogous in process to
microscopic visualization of cellular pathology patterns
represented in histologic microscopy sections. It is a rare
occurrence for a whole gel to display aberrations unless
there has been a problem with the sample preparation.
This aspect of 2DE gel performance is similar to encountering a small fold or tissue separation near one area of a
microtome section. To account for this, we include a QC
step for determining whether all of the spots in a series
of multiple isoforms appear in correct register. The con-
tinued development of specialized diagnostic indications
for serum and urine 2DE will require appropriate procedures for QC of the method, examples of which are
provided here.
Acknowledgments. The authors thank Drs. Norman G. Anderson, N.
Leigh Anderson, N. W. Cho, C. S. Giometti, A. Hirata (deceased), C.
Lee, J. Taylor, and Y. C. Tsai for their advice, encouragement, and ongoing collaborative support. RPSP was obtained from the College of
American Pathologists; additional lots of pre-release RPSP were the kind
gift of Mr. T. Snyder, Incstar (formerly Atlantic Antibodies), the manufacturer.
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