matrigel: a complex protein mixture required for optimal growth of cell culture

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

2 Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of WesternOntario, London, ON, Canada

Received: November 12, 2009

Revised: January 7, 2010

Accepted: January 20, 2010

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.

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

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 condi-

tions, 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,

Abbreviations: AS, ammonium sulfate; ECM, extracellular

matrix; GFR, growth factor reduced; GO, gene ontology; IE-MS,

iterative exclusion-MS; MW, molecular weight

Correspondence: Dr. Gilles A. Lajoie, Don Rix Protein Identifi-

cation Facility, Department of Biochemistry, Schulich School of

Medicine and Dentistry, University of Western Ontario, London,

Ont., N6A 5C1 Canada

E-mail: [email protected]

Fax: 11-519-661-3954

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1886 Proteomics 2010, 10, 1886–1890DOI 10.1002/pmic.200900758

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 undif-

ferentiated 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

standard and GFR Matrigel (Supporting Information

Fig. 2B). Analysis of each SEC fraction with one-dimen-

sional SDS-PAGE (1D-SDS-Gel) gave gels with very poor

resolution, and showed minute amounts of low–molecular-

weight (MW) proteins, as determined by Coomassie blue

staining method (Supporting Information Fig. 2A). Never-

theless, fractions resulting from SEC runs were digested in-

solution 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.

Proteomics 2010, 10, 1886–1890 1887


to an in-solution trypsin digestion. The pellet from 15% AS

precipitation of the standard matrix was used as the in-

house 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 exclusion-

MS (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 resul-

ted in the identification of 3508 unique peptides for stan-

dard and 3341 for GFR Matrigel (Fig. 1C). Combination of

SEC and AS precipitation data sets resulted in the identifi-

cation 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-

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 fractio-

nation 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 frac-

tionation 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.

1888 C. S. Hughes et al. Proteomics 2010, 10, 1886–1890


files (Fig. 2A, Supporting Information Table 1). Upon

merging of the standard and GFR Matrigel data, 125 addi-

tional 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 fibro-

blast growth factor, epidermal growth factor, insulin-like

growth factor 1, transforming growth factor beta, platelet-

derived 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 Infor-

mation 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 identifica-

tions.

IE-MS analysis of samples prepared using SEC separa-

tion and AS precipitation methods revealed a total of 1302

unique protein and 9565 peptide identifications from stan-

dard 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

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 precipita-

tion 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 stan-

dard 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, Support-

ing Information Tables 6–8). This implies that although

structurally important proteins such as laminin constitute

Figure 3. A comparative analy-

sis 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) Biologi-

cal process, (C) cellular locali-

zation, and (D) molecular

function GO assignments.

Proteomics 2010, 10, 1886–1890 1889


the bulk of what is being extracted from the Englebreth-

Holm-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 prepara-

tion. 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 GraduateScholarship doctoral award. This work was supported in part bya grant from the National Science and Engineering Council(NSERC) to G. A. L. The authors are also grateful to JonathanMeyer for helpful discussions and suggestions, and to AmeliaNuhn for manuscript editing.

The authors have declared no conflict of interest.

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matrigel: a complex protein mixture required for optimal growth of cell culture

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