This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.
Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.
Fibrosis is a hallmark of many diseases. Fibroblasts proliferate and differentiate towards highly synthetic phenotypes, which are associated with the exacerbated secretion and deposition of extracellular matrix (ECM)1. Excessive ECM deposition can continue, even after the initial injury has abated, leading to functional impairment. Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiac tissue2,3. They are not only structural proteins, but also matricellular proteins and proteases that contribute to the continuous remodeling and dynamic adaptation of the heart. Notably, many ECM proteins are glycosylated4. This post-translational modification (PTM) involves the addition of sugar residues to certain amino acid positions, and it affects protein folding, solubility, binding, and degradation5.
There are two main glycosylation types that occur in mammals. (1) N-glycosylation occurs at the carboxamido nitrogen of asparagine residues (Asn) within the consensus sequence Asn-Xaa-Thr/Ser, where Xaa is any amino acid except for proline. (2) In O-glycosylation, sugar residues attach to serine and threonine residues (Ser, Thr) or, to a much lesser extent, to hydroxyproline and hydroxylysine. While O-glycosylation can occur in a variety of protein groups, N-glycosylation is restricted to secreted proteins or extracellular domains of membrane proteins5. This makes N-glycosylation an attractive target when studying the ECM.
Proteomics sets a new standard for the analysis of protein changes in disease. Thus far, most proteomics studies have been focused on intracellular proteins6. This is mainly due to the following reasons. First, abundant intracellular proteins hamper the identification of scarce ECM components. This is particularly crucial in cardiac tissue, in which mitochondrial and myofilament proteins account for a large proportion of the protein content7. Second, integral ECM proteins are heavily cross-linked and difficult to solubilize. Lastly, the presence of abundant PTMs (i.e., glycosylation) alters the molecular mass, charge, and electrophoretic properties of peptides, affecting both the separation and the identification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Over recent years, we have developed and improved a sequential extraction and enrichment method for ECM proteins that is compatible with subsequent mass spectrometry (MS) analysis. The strategy is based on sequential incubations.
The first step is performed with NaCl, an ionic buffer that facilitates the extraction of ECM-associated and loosely bound ECM proteins, as well as newly synthesized ECM proteins. It is detergent free, non-denaturing, non-disruptive of cell membranes, and amenable for further biochemical assays8. Then, decellularization is achieved with sodium dodecyl sulfate (SDS). At this step, a low SDS concentration ensures membrane destabilization and the release of intracellular proteins whilst preventing the disruption of the more soluble non-integral ECM components. Finally, ECM proteins are extracted with a guanidine hydrochloride buffer (GuHCl). GuHCl is effective in extracting heavily cross-linked proteins and proteoglycans from tissues such as tendons9, cartilage10, vessels11,12,13 and the heart2,3. We applied this biochemical fractionation, in combination with LC-MS/MS, to explore ECM remodeling in cardiovascular disease2,3,11,12,13,14. Recent advances in MS include novel fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct analysis of intact glycopeptides3,15.
Here we describe a methodology to prepare ECM for MS analysis that allows for the analysis of protein composition, the identification of glycosylation sites, and the characterization of glycan forms. Compared to previous analyses of ECM glycosylation16, this methodology allows for the direct assessment of compositional changes in glycosylation profiles in a site-specific manner using MS. We have applied this methodology to cardiovascular tissues. However, it can also be applied, with minor modifications, to the study of the ECM in other tissue specimens and can provide unprecedented insights into ECM biology.
The study was approved by the Wandsworth Local Research Ethics Committee (reference number: 06/Q0803/37) and received institutional approval from the research and development office. All patients gave written informed consent.
1. Extraction of Extracellular Matrix Proteins
NOTE: The human atrial tissues used for these experiments were obtained from the atrial appendages during cardiopulmonary bypass, just after the cardioplegic arrest of the heart. All samples were collected at St George's Hospital, London, UK. All tissue samples must be frozen at -80 °C. Do not use samples preserved with fixatives, such as paraformaldehyde, that cross-link proteins.
2. Protein Quantification and Precipitation
NOTE: Due to the presence of detergents, the SDS buffer is incompatible with direct protein quantification based on measurements of absorbance at 280 nm. To ensure reproducible quantification, use colorimetric assays for all protein extracts17.
3. Sequential Deglycosylation for the Assessment of N-glycosylation Site Occupancy
4. In-solution Trypsin Digestion
NOTE: This step should be carried out for both non-deglycosylated (i.e., used for direct glycopeptide analysis) and deglycosylated samples (i.e., used for the assessment of glycan occupancy).
5. Peptide Cleanup Using C18 Columns
NOTE: The removal of interfering contaminants from the peptide mixture after digestion reduces ion suppression and improves signal-to-noise ratios and sequence coverage. This step should be carried out for both non-deglycosylated and deglycosylated samples.
6. Labeling with TMT (for Direct Glycopeptide Analysis Only)
7. Glycopeptide Enrichment
8. Mass Spectrometry Analysis
A schematic workflow of the protocol is provided in Figure 1.
ECM extraction protocol
The efficiency of the extraction can be monitored by running aliquots form each extract on Bis-Tris acrylamide gels and using silver staining for visualization. Figure 2A shows the complementarity of the NaCl, SDS and GuHCl extracts after sequential extraction. This QC allows for identification of potential issues with sample quality such as excessive protein degradation. After extraction, ECM glycoproteins are abundant in the GuHCl extracts (Figure 2B).
Deglycosylation
To assess the efficiency of deglycosylation, a non-deglycosylated control should be run in parallel. Deglycosylation times have to be suitable to achieve a complete and homogeneous removal of sugar residues, as exemplified in Figure 3A. Figure 3B shows a representative example of samples efficiently deglycosylated by the addition of enzymes for GAG removal and deglycosylation enzymes that target smaller N- and O-linked oligosaccharides.
Glycoproteomics
The protocol for assessment of the occupancy of NxT/S sequons improves protein sequence coverage for ECM glycoproteins after MS (Figure 3C) and allows for an initial screening of the presence of glycoproteins. This helps to reduce the search time for glycopeptides, as databases can be customized to contain previously identified glycoproteins. HCD-ETD fragmentation is used for identification and compositional characterization of oligosaccharides attached to ECM glycoproteins. Figure 4A displays a representative spectrum obtained for a peptide labeled with 18O after deglycosylation (indirect glycopeptide analysis). Figure 4B is a representative example of a spectrum obtained after analysis of intact glycopeptides from ECM extracts (direct glycopeptide analysis).
Figure 1: Method Overview. (A) After sequential enrichment for ECM proteins, LC-MS/MS analyses are performed on the deglycosylated extracts. (B) Alternatively, non deglycosylated ECM extracts are further enriched for glycopeptides. Please click here to view a larger version of this figure.
Figure 2: Extraction of ECM Proteins. (A) The 3 different extracts from the sequential extraction procedure ("English Quickstep") are complementary in their protein content. While SDS extracts are enriched in intracellular proteins, GuHCl extracts contain the majority of ECM proteins. Successful fractionation is visualized by the different silver staining pattern. (B) ECM proteins such as the small leucine-rich proteoglycans decorin, biglycan and mimecan are predominantly detected in the GuHCl extracts, with little presence in the SDS and NaCl extracts. Please click here to view a larger version of this figure.
Figure 3. Analysis of Glycosylation. (A) Appropriate incubation times are required for complete deglycosylation. The example shows the effect of incubation time during removal of glycosaminoglycan chains from the glycoprotein decorin. (B) ECM glycoproteins are decorated with large and repetitive glycosaminoglycan chains and short and diverse N- and O-linked oligosaccharides. Lane 1 on each of the immunoblots represents untreated cardiac extracts. Lane 2 contains extracts treated with enzymes that digest glycosaminoglycans. Samples in lane 3 contain, in addition, enzymes for the removal of N- and O-linked oligossacharides. Galectin-1 is not glycosylated, hence there is no shift in protein size. Adapted from Lynch M, et al.4 (C) In LC-MS/MS analysis, samples treated with PNGase-F in the presence of H218O achieve better sequence coverage (%, on the right side) compared to non-deglycosylated samples. Dark green areas represent sequence coverage by LC-MS/MS. The red, dotted lines represent glycosites, with numbers indicating their amino acid position. Detection of glycosylation of decorin at position Asn211 (N, highlighted in bold) is shown in detail as an example in Figure 4. Please click here to view a larger version of this figure.
Figure 4. Glycopeptide Analysis by MS. (A) Using a shotgun proteomics approach on ECM enriched extracts, glycopeptides can be identified by the presence of deamidated asparagines within NxT/S sequons and labeled with 18O. The example shows a HCD MS/MS spectrum for a peptide of decorin containing the previously glycosylated Asn211. The data obtained can be used to create a customized database of ECM glycoproteins. (B) HCD-ETD fragmentation is used to analyze the glycopeptide enriched ECM extract. The ETD MS/MS spectrum allows the characterization of glycan composition. Please click here to view a larger version of this figure.
A. Stock solutions | |
DTT (Dithiotreitol, C4H10O2S2) | 100 mM DTT in ddH2O.1 |
EDTA (Ethylenediaminetetraacetic acid, C10H16N2O8) | 250 mM EDTA in ddH2O, pH 8.0. |
GuHCl (Guanidine hydrochloride, CH6ClN3) | 8 M GuHCl in ddH2O. |
IAA (Iodoacetamide, C2H4INO) | 500 mM IAA in ddH2O.1,2 |
Na acetate (Sodium acetate, C2H3NaO2) | 1 M Na acetate in ddH2O, pH 5.8. |
NaCl (Sodium chloride, NaCl) | 1 M NaCl in ddH2O. |
Na phosphate dibasic (Disodium phosphate, Na2H2PO4) | 1 M Na phosphate dibasic in ddH2O, pH 6.8. |
SDS (Sodium dodecyl sulfate, NaC12H25SO4) | 1% SDS (35 mM) in ddH2O.3 |
TFA (Trifluoroacetic acid, C2HF3O2) | 10% TFA (1.2 M) in ddH2O. |
TEAB (Triethylammonium bicarbonate, C7H17NO3) | 1M TEAB in in ddH2O, pH 8.5 |
Thiourea (Thiourea, CH4N2S) | 3 M thiourea in ddH2O. |
Tris-HCl (Tris-hydrochloride (NH11C4O3[HCl]) | 100 mM Tris–HCl in ddH2O, pH 7.5. |
Urea (Urea, CH4N2O) | 9 M urea in ddH2O. |
B. Reaction buffers | |
C18 clean-up equilibration buffer | 1% ACN, 0.1% TFA in ddH2O |
C18 clean-up column wash buffer | 80% ACN, 0.1% TFA in H2O |
C18 clean-up elution buffer | 50% acetonitrile, 0.1% TFA in ddH2O |
Deglycosylation Buffer (4x) | 600 mM NaCl and 200 mM Na phosphate in ddH2O, pH 6.8. |
GuHCl buffer4 | 4 M guanidine hydrochloride, 50 mM Na acetate and 25 mM EDTA in ddH2O, pH 5.8. Add 1:100 (v:v) of cocktail of proteinase inhibitors before use. |
NaCl buffer4 | 0.5 M NaCl, 10 mM Tris-HCl and 25 mM EDTA in ddH2O, pH 7.5. Add 1:100 (v:v) of cocktail of proteinase inhibitors before use. |
PBS (1x) | 1.7 mM KH2PO4, 5 mM Na2HPO4, 150 mM NaCl, pH 7.4. Add 25 mM EDTA and 1:100 (v:v) of cocktail of proteinase inhibitors before use. |
Sample buffer (4x) | 100 mM Tris, 2% SDS, 40% Glycerol, 0.02% bromophenol blue in ddH2O, pH 6.8. Add 10% ß-mercaptoethanol before use. |
SDS buffer4 | 0.1 % SDS and 25 mM EDTA in ddH2O. Add 1:100 (v:v) of cocktail of proteinase inhibitors before use. |
C. Enzymes | |
Chondroitinase ABC5 | 0.5 U/mL in deglycosylation buffer (1x) |
Keratanase5 | 0.1 U/mL in deglycosylation buffer (1x) |
Heparinase II5 | 0.1 U/mL in deglycosylation buffer (1x) |
α2-3,6,8,9-Neuraminidase (sialidase)5 | 0.025 U/mL in deglycosylation buffer (1x) |
β1,4-Galactosidase5 | 0.015 U/mL in deglycosylation buffer (1x) |
β-N-Acetylglucosaminidase5 | 0.25 U/mL in deglycosylation buffer (1x) |
Endo-α-N-acetylgalactosaminidase (O-glycosidase)5 | 0.013 U/mL in deglycosylation buffer (1x) |
PNGase-F(N-glycosidase-F)6 | 50 U/mL in H218O |
Trypsin | 0.01 µg/µL in TEAB buffer |
Table NOTES. | |
1 Keep stock solution frozen at -20 °C. | |
2 IAA should be kept protected from light. | |
3 SDS readily crystallizes at < 20 °C. In order to facilitate solubilization of 1% SDS (stock solution), warm the buffer under hot tap water. | |
4 Extraction buffers can be stored at RT. Add broad-spectrum cocktail of proteinase inhibitors as indicated before use. | |
5 These enzymes should be added during the first deglycosylation step. | |
6 PNGase-F should be only added during the second deglycosylation step. |
Table 1: Stock Solutions, Reaction Buffers and Enzymes. This table lists the composition of each stock solution and reaction buffer required for the extraction and subsequent processing (including enzymatic digestions) of cardiac ECM proteins prior to MS analysis.
This proteomics protocol has been optimized over the last few years in our laboratory. Here, we used cardiac tissue, but only minor adjustments may be required for its application to other tissues. For example, the extraction protocol needs to take the cellularity of the tissue into consideration. Cardiac tissue is highly cellular compared to vascular tissue. When using vascular tissue, the SDS concentration can be lower (i.e. 0.08%) and the decellularization time is shorter (i.e. 4 h)11,12,13. The use of deglycosylation enzymes is crucial for LC-MS/MS analysis of ECM composition. However, incubation times need to be adjusted for different tissue types. For example, heparinase II required extended incubation times at 25 °C when using samples such as skin, which are rich in basement membrane proteins (e.g. agrin, perlecan) (data not shown). Direct glycopeptide analysis can be performed on conditioned media from cells in culture15. Enrichment steps may not be required for the analysis of this simplified subproteome. Similar to GuHCl extracts, NaCl extracts are also amenable for glycoproteomics analysis with minor modifications. Other extraction protocols for enrichment of ECM proteins can be adapted to characterize ECM glycopeptides19,20.
Glycosylation is the most complex PTM5. Indirect identification of glycopeptides is achieved by the detection of deamidated Asn with incorporated 18O at a NxT/S sequon. Deamidated Asn at other positions may represent false positives. Likewise, N-glycosylation must be considered in the context of protein ontologies: intracellular proteins containing a NxT/S sequon will not be glycosylated but might give rise to false positives. As current search algorithms do not allow for the screening of PTMs at pre-determined sequences only (i.e. Asn at NxT/S), manual filtering of the data is required. Identification of presence/absence of glycosylation at these positions can be compared between disease and control samples. There is no enzyme equivalent to PNGase F for O-deglycosylation (i.e. introducing a mass shift on threonine or serine). Therefore, the identification of O-glycosylation is restricted to direct glycopeptide analysis. Direct glycopeptide analysis is used to obtain compositional information of sugars attached to proteins, but does not provide structural information of the glycan. Moreover, the glycan composition is the result of glycan synthesis and processing after secretion.
Our 3-step extraction method for ECM proteins ("English Quickstep")6 has allowed characterizing the ECM in a variety of cardiovascular tissues. Fractionation of the tissue into several extracts is required to obtain a simplified ECM proteome as discussed elsewhere6. Intracellular proteins would otherwise contribute to an excessive dynamic range of protein abundances within the extracts that would hinder identification of less abundant ECM proteins. Moreover, intracellular proteins carry O-glycosylations5 that would complicate ECM glycopeptide enrichment and subsequent MS analysis. Other authors applied similar extraction methodologies to characterize for example lung21 and cartilage tissues10, however they did not pursue the analysis of glycosylation. Previous analysis of glycosylation focused on identification of glycosites only, require removal of the glycan from the protein core, and cannot assess O-glycosylation22,23. Lectin arrays and chemical enrichment are available for assessment of glycan types on biological samples based on their binding specificity, but these techniques cannot assign glycan types to specific proteins24 nor can they assess glycosylation sites.
Initially, we used gel electrophoresis prior to LC-MS/MS of ECM proteins. Although gel separation is useful in obtaining simplified protein fractions amenable to LC-MS/MS analysis, the latest instruments offer faster scan speeds. Thus, the electrophoretic separation step can be omitted. This provides an additional advantage as large ECM proteins, which are retained on top of the gel, are analyzed more efficiently. However, information regarding the Mw of the intact proteins is lost. The evaporation step prior to PNGase F deglycosylation ensures complete removal of regular H2O to minimize false negatives. Sugar residues (i.e. variable glycan masses) interfere with the separation by LC and compromise subsequent peptide identification by MS/MS. A pan-deglycosylation protocol is also recommended for proteomics analysis of ECM proteins not focused on glycosylation.
Proteomics can provide unprecedented insights into the ECM. Beyond structural support, glycans attached to the ECM are essential for host-pathogen interaction, cell-cell communication and the immune response25, i.e. allograft rejection after organ transplantation. Glycoproteomics will be an essential tool in glycobiology.
The authors have nothing to disclose.
JBB is a Career Establishment Fellow in the King’s British Heart Foundation Centre. MM is a Senior Fellow of the British Heart Foundation (FS/13/2/29892). The study was supported by an excellence initiative (Competence Centers for Excellent Technologies – COMET) of the Austrian Research Promotion Agency FFG: “Research Center of Excellence in Vascular Ageing – Tyrol, VASCage” (K-Project number 843536) and the NIHR Biomedical Research Center based at Guy’s and St. Thomas’ National Health Service Foundation Trust and King’s College London in partnership with King’s College Hospital.
A. Chemicals | |||
Acetonitrile, MS-grade (ACN, C2H3N) | Thermo Scientific | 51101 | 5.2-5.8, 6.2, 7.11, Supp 2, 3, 4 |
Cocktail of proteinase inhibitors | Sigma-Aldrich | P8340 | 1.3, 1.4.1, 1.5.1, 1.6.1 |
Disodium phosphate (Na2H2PO4) | Sigma-Aldrich | S7907 | 3.1 |
Dithiotreitol (DTT, C4H10O2S2) | Sigma-Aldrich | D0632 | 4.3 |
Ethylenediaminetetraacetic acid (EDTA, C10H16N2O8) | Sigma-Aldrich | E9884 | 1.3, 1.4.1, 1.5.1, 1.6.1 |
Ethanol (C2H6O) | VWR | 437433T | 2.2.1 |
Guanidine hydrochloride (GuHCl, CH6ClN3) | Sigma-Aldrich | G3272 | 1.6.1 |
Glycerol (C3H8O3) | Acros organics | 158920025 | Suppl 1.1 |
H2O LC-MS Cromasolv | Sigma-Aldrich | 39253-1L-R | Throughout the protocol |
H218O | Taiyo Nippon Sanso | FO3-0027 | 3.5 |
Hydroxylamine (HA, H3NO) | Sigma-Aldrich | 467804 | 6.4 |
Iodoacetamide (IAA, C2H4INO) | Sigma-Aldrich | A3221 | 4.4 |
Phosphate-buffered Saline (PBS), 10X | Lonza | 51226 | 1.3 |
Sodium acetate (C2H3NaO2) | Sigma-Aldrich | S7545 | 1.6.1 |
Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | 1.4.1, 3.2 |
Sodium dodecyl sulfate (SDS, NaC12H25SO4) | Sigma-Aldrich | 466143 | 1.5.1 |
Triethylammonium bicarbonate (TEAB, C7H17NO3) | Sigma-Aldrich | 11268 | 4.7, 6.1 |
Trifluoroacetic acid (TFA, C2HF3O2) | Sigma-Aldrich | T62200 | 4.8, 5.2-5.8, 7.11, Supp 2, 3 |
Thiourea (CH4N2S) | Sigma-Aldrich | T8656 | 4.2 |
Tris-hydrochloride (Tris-HCl, NH11C4O3[HCl]) | Sigma-Aldrich | T3253 | 1.4.1, Suppl 1. |
Urea (CH4N2O) | Sigma-Aldrich | U1250 | 4.2 |
Name | Company | Catalog Number | Comments |
B. Enzymes | |||
α2-3,6,8,9-Neuraminidase (Sialidase) | EDM Millipore | 362280 (KP0012) | 3.1 |
β1,4-Galactosidase | EDM Millipore | 362280 (KP0004) | 3.1 |
β-N-Acetylglucosaminidase | EDM Millipore | 362280 (KP0013) | 3.1 |
Chondroitinase ABC | Sigma-Aldrich | C3667 | 3.1 |
Endo-α-N-acetylgalactosaminidase (O-glycosidase) | EDM Millipore | 362280 (KP0011) | 3.1 |
Heparinase II | Sigma-Aldrich | H6512 | 3.1 |
Keratanase | Sigma-Aldrich | G6920 | 3.1 |
PNGase-F (N-Glycosidase F) | EDM Millipore | 362280 (KP0001) | 3.5 |
Trypsin | Thermo Scientific | 90057 | 4.7 |
Name | Company | Catalog Number | Comments |
C. Reagent kits | |||
30 kDa MWCO spin filters | Amicon, Millipore | 10256744 | 5.9, Suppl 2 |
Macro SpinColumn C-18, 96-Well Plate | Harvard Apparatus | 74-5657 | 5.1 |
NuPAGE Novex BisTris Acrylamide Gels | Thermo-Scientific | NP0322PK2 | Suppl 1 |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23227 | 2.1.3 |
ProteoExtract Glycopeptide Enrichment Kit | Merk Millipore | 72103 | 7 |
Tandem mass tag 0 (TMT0) | Thermo Scientific | 900067 | 6.2, 6.3 |
Name | Company | Catalog Number | Comments |
D. Equipment and software | |||
Acclaim PepMap100 C18 Trap, 5mm x 300µm, 5µm, 100Å | Thermo Scientific | 160454 | Suppl 3, 4 |
Acclaim PepMap100 C18, 50cm x 75µm, 3µm, 100Å | Thermo Scientific | 164570 | Suppl 3 |
Byonic Search Engine | Protein Metrics | Version 2.9.30 | Suppl 5 |
Dionex UltiMate 3000 RSLCnano | Thermo Scientific | n/a | Suppl 3, 4 |
EASY-Spray Ion Source | Thermo Scientific | ES081 | Suppl 4 |
EASY-Spray PepMap RSLC C18, 50cm x 75µm, 2μm, 100Å | Thermo Scientific | ES803 | Suppl 4 |
Mascot Search Engine | Matrix Science | Version 2.3.01 | Suppl 3 |
Orbitrap Fusion Lumos Tribrid Mass Spectrometer | Thermo Scientific | IQLAAEGAAPFADBMBHQ | Suppl 4 |
Proteome Discoverer Software | Thermo Scientific | Version 2.1.1.21 | Suppl 3, 5 |
Picoview Nanospray Source | New Objective | 550 | Suppl 3 |
Q Exactive HF Mass Spectrometer | Thermo Scientific | IQLAAEGAAPFALGMBFZ | Suppl 3 |
Savant SpeedVac Concentrator | Thermo Scientific | SPD131DDA | 2.2.2, 3.4, 4.6, 5.7, 6.5, 7.11 |
Scaffold Software | Proteome Software | Version 4.3.2 | Suppl 3 |