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1886

DOI 10.1002/pmic.200900758

Proteomics 2010, 10, 1886–1890

TECHNICAL BRIEF

Matrigel: A complex protein mixture required for optimal
growth of cell culture
Chris S. Hughes1, Lynne M. Postovit2 and Gilles A. Lajoie1
1

2

Don Rix Protein Identification Facility, Department of Biochemistry, Schulich School of Medicine and Dentistry,
University of Western Ontario, London, ON, Canada
Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western
Ontario, London, ON, Canada

Numerous cell types require a surface for attachment to grow and proliferate. Certain cells,
particularly primary and stem cells, necessitate the use of specialized growth matrices along
with specific culture media conditions to maintain the cells in an undifferentiated state. A
gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel
is commonly used as a basement membrane matrix for stem cells because it retains the stem
cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and
therefore can produce a source of variability in experimental results. In this study, we present
an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method
coupled with fractionation protocols that involve ammonium sulfate precipitation, size
exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low
mass and abundance components of Matrigel illustrates the utility of this method for the
analysis of the extracellular matrix, as well as the complexity of the matrix itself.

Received: November 12, 2009
Revised: January 7, 2010
Accepted: January 20, 2010

Keywords:
Cell biology / Protein profile / Protein digest / Quadrupole time of light /
Tandem mass spectra / Tryptic digest

The in vivo and in vitro extracellular matrix (ECM) is known
to play an important role in numerous decisions that direct
cell fate and behavior [1–4]. Typical ECM proteins include
laminin, collagens, glycoproteins, and proteoglycans [5, 6].
The main function of the ECM is to support the growth and
maintenance of a variety of cells. In vitro growth matrices
can be a variety of materials, such as chemically treated
culture dish plastic, or layers of deposited protein. Other
matrices attempting to mimic the 3-D in vivo environment
have also been developed [7]. Often these matrices are very
Correspondence: Dr. Gilles A. Lajoie, Don Rix Protein Identification Facility, Department of Biochemistry, Schulich School of
Medicine and Dentistry, University of Western Ontario, London,
Ont., N6A 5C1 Canada
E-mail: glajoie@uwo.ca
Fax: 11-519-661-3954
Abbreviations: AS, ammonium sulfate; ECM, extracellular
matrix; GFR, growth factor reduced; GO, gene ontology; IE-MS,
iterative exclusion-MS; MW, molecular weight

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

simple, consisting of a mixture of purified proteins such as
collagen and laminin. Matrices such as these, which contain
only major ECM proteins, are not applicable to all cell types.
ECM mixtures extracted from living cells are commonly
used to grow cells that are more sensitive to culture conditions, likely due to the inclusion of critical growth factors
and cytokines. The most widely utilized example of this
is the cell culture matrix commercialized as Matrigel
(BD Biosciences, Mississauga, Canada) [8, 9].
Matrigel is an assortment of ECM proteins that have been
extracted from Englebreth-Holm-Swarm tumors in mice
[8–10]. Matrigel, which primarily consists of laminin,
collagen IV, and enactin, is considered to be a reconstituted
basement membrane preparation. A previous publication
advised caution when drawing conclusions based on the
changes in cellular activity while using Matrigel [11]. This
recommendation was based on the detection of several
growth factors in standard Matrigel through the use of
immunoassays; these included basic fibroblast growth
factor, epidermal growth factor, insulin-like growth factor 1,
www.proteomics-journal.com

Proteomics 2010, 10, 1886–1890

transforming growth factor beta, platelet-derived growth
factor, and nerve growth factor [11]. The growth factor
reduced (GFR) is a version of Matrigel that has been
modified to reduce abundance levels of these growth factors
[11].
One important application of Matrigel is for the growth
of human embryonic stem cells. Matrigel is used to mimic
the ECM in cancer and stem cell culture, presumably by
replicating cell–ECM interactions. Matrigel has been shown
to be an optimal matrix for culture of stem cells because of
its ability to maintain self-renewal and pluripotency. Exactly
how Matrigel helps the stem cells to remain in an undifferentiated state, remains poorly understood. A contributing
factor to this lack of understanding is the difficulty in the
analysis of the ECM. Because of the widespread use of
Matrigel in cell culture and cancer research, there is critical
need to determine its composition.
The main protein components of Matrigel, laminin
(800 000 Da), collagen IV (540 000 Da), and enactin
(158 000 Da), are all significantly larger than the average size
of most protein growth factors (o45 000 Da). In SEC runs of
neat Matrigel samples, we were unable to obtain good
resolution. We observed a large peak early due to the
more rapid elution of the large proteins from both the

1887
standard and GFR Matrigel (Supporting Information
Fig. 2B). Analysis of each SEC fraction with one-dimensional SDS-PAGE (1D-SDS-Gel) gave gels with very poor
resolution, and showed minute amounts of low–molecularweight (MW) proteins, as determined by Coomassie blue
staining method (Supporting Information Fig. 2A). Nevertheless, fractions resulting from SEC runs were digested insolution with trypsin before analysis by LC-MS with a Q-ToF
Ultima (Waters, Milford, MA).
To profile the low MW and abundance components of
Matrigel, a method of depleting the large amount of high
MW components, such as laminin, was needed. Due to the
limited separation and loading capacity provided by
1D-SDS-Gels, and the poor resolution of SEC, we adapted a
two-step ammonium sulfate (AS) precipitation protocol to
fractionate both the standard and GFR Matrigel (Supporting
Information Fig. 1). AS precipitation is not commonly used
in proteomics but was used for the depletion of standard
Matrigel to generate the GFR variety [11]. The two-step AS
procedure utilized here first precipitates protein at 15% AS,
followed by a 90% treatment of the resultant supernatant for
full protein extraction.
The 15% AS step gave a precipitate that was further
fractionated by SEC to yield 15 fractions that were subjected

Figure 1. IE analysis of the Matrigel samples. (A) The total number of proteins and peptides identified in each round of IE analysis.
Peptides and proteins identified for the two varieties of Matrigel tested, standard and GFR, from all fractionation protocols. The number of
protein and peptide hits is given above each set of exclusion bars in the format protein] (peptide]). Standard values are in normal font,
whereas GFR values are in italics. Protein lists for exclusions can be found in Supporting Information Table 9. (B) The number of peptide
identifications across six exclusion rounds for laminin/enactin. (C) The number of unique peptide identified in sample sets for each type of
Matrigel, as well as combined, for different fractionation methods used in sample preparation. LC-MS represents a single injection for
each sample, whereas IE-MS represents multiple injections with exclusion lists applied during data acquisition.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.proteomics-journal.com

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C. S. Hughes et al.

to an in-solution trypsin digestion. The pellet from 15% AS
precipitation of the standard matrix was used as the inhouse prepared GFR Matrigel. The supernatants of both the
standard Matrigel and GFR from the first AS precipitation
step were precipitated at 90% AS. The resulting pellets were
separated on a 1D-SDS-Gel. These gels contained visible
amounts of low MW proteins after staining (Supporting
Information Fig. 1A). From these gels, ten bands were cut,
digested with trypsin, and subjected to iterative exclusionMS (IE-MS) analysis [12]. After removal of the pellet, 90%
AS supernatant solutions were tested for protein content
using the Bradford assay to ensure full protein extraction
(data not shown).
The LC-MS analysis of the SEC fractions from standard
Matrigel resulted in the identification of 1012 unique
peptides. The same analysis on GFR Matrigel SEC fractions
resulted in the identification of 872 unique peptides
(Fig. 1c). As expected, the majority of the identifications
were very similar with 768 peptides in common, with
the exception of 244 found only in standard and 104
detected only in GFR. LC-MS analysis of the pellets from
90% AS precipitation after 1D-SDS-Gel fractionation resulted in the identification of 3508 unique peptides for standard and 3341 for GFR Matrigel (Fig. 1C). Combination of
SEC and AS precipitation data sets resulted in the identification of 3842 unique peptides for standard and 4025 for
GFR Matrigel.
Even after SEC fractionation and AS precipitation
followed with 1D-SDS-Gels, there was still a considerable
dynamic range to address. IE-MS allows for in-depth
analysis of the samples resulting in the identification of low-

Proteomics 2010, 10, 1886–1890

abundance proteins [11]. The IE-MS analysis of the SEC
fractions resulted in the identification of 2781 unique
peptides for the standard and 2082 for the GFR Matrigel
following four exclusion rounds (Fig. 1C). The IE-MS
analysis of pellets from 90% AS precipitation after fractionation with 1D-SDS-Gel resulted in the identification of
8964 unique peptides for standard and 7326 for GFR
Matrigel following six exclusion rounds (Fig. 1C). After
combining the IE-MS data sets from each fractionation we
observed a significant increase in the number peptides
identified with 9565, corresponding to 1302 proteins in the
standard Matrigel. This trend is also held true for the GFR
samples. After six rounds of exclusion, 9417 unique
peptides and 1246 proteins were matched. These values
represent an approximate threefold increase in the number
of identifications through the use of IE-MS with samples
from all fractionation types.
After combination of the data sets for SEC fractionation
of standard and GFR Matrigel, we identified 3669 peptides
and 515 unique proteins after IE-MS analysis (Fig. 1C,
Supporting Information Table 4). The pellets obtained from
90% AS precipitation of GFR and standard Matrigel resulted
in 12 213 peptide and 1437 unique protein identifications
after 1D-SDS-Gel fractionation and IE-MS analysis (Fig. 1C,
Supporting Information Table 5). These results illustrate the
utility of AS precipitation for the detection of the proteins in
a complex sample when used in combination with IE-MS in
comparison with SEC methods.
Combining the data from all Matrigel batches and fractionation protocols, we identified a total of 14 060 unique
peptides and 1851 unique proteins from 280 LC-MS data

Figure 2. GO assignments for the consolidated standard and GFR Matrigel data set, including all methods of fractionation. (A) Biological
process assignments, (B) cellular localization assignments, (C) molecular function assignments, with corresponding categories found on
the right of each pie graph. The number 1851 in the red circle, denotes the total number of protein identifications made across all the
Matrigel samples after data set consolidation.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.proteomics-journal.com

Proteomics 2010, 10, 1886–1890

files (Fig. 2A, Supporting Information Table 1). Upon
merging of the standard and GFR Matrigel data, 125 additional proteins (2 unique peptides) were identified due to the
combination of single peptide hits from each data set. The
major Matrigel components were found to be laminin and
enactin, with few peptides matched for collagen. In addition
to those most abundant proteins, the majority of the
peptides identified in early rounds of analysis were for
structural proteins such as actin, spectrin, tubulin, dynactin,
and filamin.
GFR Matrigel is a variant of normal Matrigel that has
decreased levels of the growth factors such as basic fibroblast growth factor, epidermal growth factor, insulin-like
growth factor 1, transforming growth factor beta, plateletderived growth factor, and nerve growth factor [11]. This is
achieved using an AS precipitation protocol, which we have
adapted to generate our own batch of GFR Matrigel for
supplemental analysis and comparison (Supporting Information Fig. 1). Analysis of GFR Matrigel samples prepared
using SEC and AS precipitation followed by in-solution or
in-gel tryptic digestion resulted in the identification of 1246
unique proteins from 9417 peptides (Fig. 3A, Supporting
Information Table 3). Through IE-MS analysis of the two
manufacturer obtained GFR Matrigel lots fractionated with
SEC and AS precipitation in triplicate, we observed only
53% batch-to-batch similarity based on protein identifications.
IE-MS analysis of samples prepared using SEC separation and AS precipitation methods revealed a total of 1302
unique protein and 9565 peptide identifications from standard Matrigel samples (Supporting Information Table 2). A
comparison of the standard Matrigel data set with GFR
revealed that there were at least 822 proteins in common
between the two Matrigel preparations (Fig. 3A). Standard
Matrigel yielded 480 other unique proteins, and the GFR

1889
Matrigel resulted in an additional 424 unique proteins. Of
the common proteins, structural proteins, such as laminin/
enactin, fibronectin, fibrinogen, dynein, and desmin,
showed a significant increase in abundance based on the
number of peptides identified in GFR Matrigel. In the
standard Matrigel, other structural proteins such as myosin
and transferrin were more abundant. In addition, numerous
intracellular proteins such as adenylate kinase and heat
shock family members also increased in standard Matrigel
(Supporting Information Table 11).
There are a number of proteins apparently unique to
either type of Matrigel. We speculate that this is due to the
presence of a large number of low-abundance proteins and
the variability of components from batch to batch and not
due to the reproducibility of the MS protocol. We should
point out that the vast majority of the apparently unique
proteins are matched with less than three unique tryptic
peptides, indicating their relative low abundance within the
sample. In addition we observed that there is an apparent
increase in the abundance of large structural proteins in
GFR Matrigel, in contrast to small intracellular species
within the standard matrix. Therefore, when AS precipitation is performed on GFR Matrigel, the supernatant fraction
is significantly less complex in comparison with that from
the standard version (Supporting Information Fig. 2). This
reduction in complexity facilitates the identification of other
low-abundance proteins in the GFR Matrigel. In the standard Matrigel, these lower abundance components are not
detected presumably because of the increased levels of low
MW protein.
Gene ontology (GO) analysis of the Matrigel data set
indicates the presence of numerous cellular proteins that
were either cytoplasmic or nuclear (Figs. 2 and 3, Supporting Information Tables 6–8). This implies that although
structurally important proteins such as laminin constitute

Figure 3. A comparative analysis between standard and GFR
Matrigel. (A) Venn diagram
with the number of protein
identifications for GFR (green)
and standard (blue) Matrigel. A
comparison between the GO
values for standard and GFR
Matrigel based on STRAP and
PIPE assignments. (B) Biological process, (C) cellular localization, and (D) molecular
function GO assignments.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.proteomics-journal.com

1890

C. S. Hughes et al.

the bulk of what is being extracted from the EnglebrethHolm-Swarm sarcoma cells, there are also numerous
intracellular proteins present. Depletion of low MW
components like growth factors using AS precipitation to
create GFR Matrigel results in the enrichment of laminin/
enactin peptides in comparison with the standard preparation. However, the lack of specificity for this method means
that numerous other matrix components are lost, as shown
through the analysis of the supernatent obtained after 15%
AS precipitation.
As noted above, Matrigel was previously reported to
contain specific growth factors [11]. With the optimized
proteomics protocol used here, we identified several growth
and transcription factors such as kruppel-like factor 6,
kruppel-like factor 15, and connective tissue growth factor.
However, the growth and transcription factors represent
only a small fraction of the proteins identified in the analysis
of the matrix. We identified numerous proteins directly
related to the binding and signaling of growth factors
(Supporting Information Fig. 3a). This in-depth proteomic
analysis of Matrigel reveals a complex and intricate mixture
of proteins consisting of structural proteins, growth factors
and their binding proteins as well as several other proteins
of roles that are not clear in cell culture. We speculate that
many of these proteins play a role in the self-renewal of
stem cells when cultured in the presence of Matrigel.
However, as a result of the significant depth of coverage we
were able to obtain with the use of AS-IE-MS methods, this
study also suggests that it will be challenging to replace
Matrigel in a variety of cell culture and experimental assays
due to its complexity.
C. S. H. is supported by a NSERC Canada Graduate
Scholarship doctoral award. This work was supported in part by
a grant from the National Science and Engineering Council
(NSERC) to G. A. L. The authors are also grateful to Jonathan
Meyer for helpful discussions and suggestions, and to Amelia
Nuhn for manuscript editing.
The authors have declared no conflict of interest.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2010, 10, 1886–1890

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