Comparative Reevaluation of FASP and Enhanced FASP Methods by

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Technical Note

Comparative Re-evaluation of FASP and Enhanced FASP Methods by LC-MS/MS Andrew JM Nel, Shaun Garnett, Jonathan M. Blackburn, and Nelson C Soares J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr501266c • Publication Date (Web): 26 Jan 2015 Downloaded from http://pubs.acs.org on February 10, 2015

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Journal of Proteome Research

Comparative Re-evaluation of FASP and Enhanced FASP Methods by LCMS/MS

Andrew JM Nel, Shaun Garnett, Jonathan M Blackburn* and Nelson C Soares* Division of Medical Biochemistry, Institute of Infectious Disease & Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South Africa.

*To whom correspondence should be addressed.

Prof. Jonathan M Blackburn Applied & Chemical Proteomics group Division of Medical Biochemistry, Institute of Infectious Disease & Molecular Medicine Faculty of Health Sciences, University of Cape Town Anzio, Observatory Cape Town 7925 South Africa Phone: +27 21 406 6071 Fax: +27 21 650 4833 e-mail: [email protected]

1 ACS Paragon Plus Environment


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Dr. Nelson C Soares Applied & Chemical Proteomics group Division of Medical Biochemistry, Institute of Infectious Disease & Molecular Medicine Faculty of Health Sciences, University of Cape Town Anzio, Observatory Cape Town 7925 South Africa Phone: +27 21 650 7607 Fax: +27 21 650 4833 e-mail: [email protected]


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Abstract Filter-Aided Sample Preparation is a proteomic technique for the preparation and on column proteolysis of proteins. Recently an enhanced FASP protocol was developed that uses deoxycholic acid (DCA) and which reportedly enhances trypsin proteolysis resulting in increases cytosolic and membrane protein representation. FASP and eFASP were reevaluated by ultra-high performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyser (Q Exactive). Although there was no difference in trypsin activity, 14099 and 13414 peptides, describing 1723 and 1793 protein groups, from Escherichia coli K12 were identified using FASP and eFASP, respectively. Characterisation of the physicochemical properties of identified peptides, showed no significant differences other than eFASP extracting slightly more basic peptides. At the protein level, both methods extracted essentially the same number of hydrophobic transmembrane helix-containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes. By employing state-of-the-art LC-MS/MS shot gun proteomics, our results indicate that FASP and eFASP showed no significant differences at the protein level. However, due to the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.



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Keywords Filtered aided sample preparation, FASP, enhanced FASP, eFASP, Q Exactive, deoxycholic acid, physicochemical, Escherichia coli K12 Abbreviations DCA-deoxycholic acid DTT- dithiothreitol eFASP- enhanced Filtered Aided Sample Preparation FASP – Filtered Aided Sample Preparation HCD – higher energy collisional dissociation HDMS – high definition mass spectrometer IAA - iodacetamide LC-liquid chromatography LTQ – linear ion trap MS – mass spectrometry MWCO – molecular weight cut off NRPs –non-redundant peptides NRPGs –non-redundant protein groups qTOF – quadrupole time-of-flight SDS – sodium dodecyl sulfate 4 ACS Paragon Plus Environment

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Introduction The filter-aided sample preparation (FASP) method is used for the purification and on-filter digestion of proteins prior to mass spectrometry based analyses.1,2 FASP was designed for the removal of detergents, chaotropes (in particular SDS), and reducing agents that were used in the extraction, solubilisation and reduction of protein disulphide bonds. In addition, FASP removes non proteinaceous components such as salts, nucleic acids, and lipids. Extracted solubilised and reduced protein samples are loaded onto a 30 000 molecular weight cut off filtration device after which detergents, lipids and small molecules are removed using a series of urea exchanges.1–3 Alkylation of reduced cysteine residues is also carried out on filter, after which protein is proteolysed on filter in the optimal buffer of the enzyme. Subsequent elution and desalting of the peptide rich solution then provides a sample ready for LCMS/MS analysis. Recently Erde et al., (2014) described an enhanced FASP (eFASP) workflow that included 0.2% DCA in the exchange, alkylation and digestion buffers. DCA was reported to increase trypsin digestion efficiency for both cytosolic and membrane proteins.4 The implementation of DCA in eFASP was based on previous reports where the sodium salt form of the surfactant successfully increased membrane protein representation in solution and in gel digest sample preparations.4–11 The non-salt form of DCA in eFASP was primarily used to avoid introduction of non-volatile sodium counter-ions that require additional desalting.4 The eFASP protocol was originally evaluated at an average mass spectrometry resolution of 9500 on a Synapt qTOF HDMS or Xevo qTOF MS.4 The success of the eFASP method was based on the identification of 252 E. coli protein groups, a twelve fold increase than that obtained using the FASP method.



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By contrast, the FASP method was originally evaluated on a LTQ-Orbitrap where 2750, 2241 and 2745 proteins were identified for HeLa cell culture, mouse brain and liver, respectively.2 Furthermore, two subsequent reports of E. coli lysates, using an LTQ Orbitrap-XL and an LTQ Orbitrap–Velos, respectively, showed that FASP outperformed non-FASP detergent removal techniques, resulting in 1058 and 1400 identified proteins in the two studies.12,13 More recently, a multi-enzyme based FASP (MED-FASP) protocol that used LysC and trypsin digestion has been reported to give approximately 2200 identified E. coli proteins per sample. This MED-FASP was evaluated on a Q Exactive MS.14 High resolution hybrid Orbitrap mass spectrometers are commonplace in proteomic analyses due to the ability to sequence and identify peptides from complex mixtures by MS/MS. The unique arrangement of selective quadrupole, high-efficiency C-trap and higher energy collisional dissociation (HCD) octopole collision cell within the Q Exactive allows fast and sensitive generation of fragment ions, enabling almost simultaneous MS and tandem MS measurements.15 There have been two reports of further optimisations of the FASP protocol in conjunction with a Q Exactive, involving refinement of the MS scanning rate - more specifically alteration to the HCD scanning rate - and coupling of a ultra-high pressure liquid chromatography (uHPLC) to the mass spectrometer, which have led to identification of approximately 4000 yeast proteins per analysed sample.16,17 Here we carried out an independent comparison between FASP and eFASP methods using proteins extracted from E. coli K12 and analysed on a uHPLC-Q Exactive mass spectrometry system operating at a resolution in MS1 of 35 000 at 200 m/z and in MS/MS of 17 500 at 200 m/z. This technical note explores the effectiveness of FASP and eFASP methods, based on both peptide yields and protein coverage. The properties of the peptides and proteins identified using either method were also characterised along with those generated from an in


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silico digest of E.coli K12 proteome. In addition, we modified our stage tip C18 desalting method to remove DCA.

Experimental section Preparation of E. coli cell extracts: An overnight culture of E. coli K12 strain M182 (Coli Genetic Stock Center #6646) was used to inoculate 200 mL of Luria Bertrani broth. Bacterial cells were incubated at 37oC with 200 rpm shaking and harvested at a mid-log phase at an optical density at 600 nm of 0.4. Cells were pelleted at 10 000 g and cells were washed three times with phosphate buffered saline pH 7.5. Harvested cell pellets were resuspended in RIPA buffer (150 mM NaCl, 50 mM triethylammonium bicarbonate, 1% SDS, 0.5% DCA, pH 8) and lysed using three cycles of heat denaturation at 95oC for 10 min and freezing at 80oC for 10 minutes. Cell debris was removed at 15 000 x g for 30 min and clarified protein extract concentrations were estimated using bicinchoninic acid (BCA) assay (Pierce, Thermo Scientific). Cysteine bonds were reduced with 100 mM DTT for 1 hr at room temperature. FASP and eFASP: All buffer exchanges were carried out by centrifugation at 14 000 x g for 15 min. 265 µL containing 200 µg of protein extract was transferred into a 500 µL Ultracel 30 000 MWCO centrifugal unit (Amicon Ultra, Merck) in triplicate for each method. Protein extracts were buffer exchanged with 3 rounds of 200 µL of UT buffer (8 M urea, 0.1 M Tris, pH 8.5). Alkylation of reduced cysteine bonds was carried out by incubation in the dark for 20 min in 200 µL UT buffer containing 0.05 M iodacetamide (Sigma). Two 200 µL UT buffer exchanges were used to remove the alkylating agent, followed by three buffer exchanges with 100 µL ABC buffer (50 mM ammonium bicarbonate buffer, pH 8). 40 µL of ABC buffer containing 1:50 ratio of sequencing grade modified trypsin (Promega) to amount of protein was added to the retentate. In the case of eFASP, all buffers used to this stage



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contained 0.2% DCA. Proteolysis was carried out at 37oC for 18 hrs in a wet chamber. Three rounds 40 µL of ABC buffer was used to elute the peptide rich solution. Desalting: Peptide rich eluate obtained for each FASP and eFASP sample was desalted using a home-made stage tip containing EmporeTM Octadecyl C18 solid phase extraction disk (Supelco).18 Activation, equilibration and peptide wash and elution were all carried out using centrifugation at 5000 x g for 5 min. Activation and equilibration of the C18 disk was carried out using three rinses with 80% acetonitrile (ACN) followed by three rinses with 2% ACN, respectively. Peptide rich solution was loaded onto the disk and centrifuged. Desalting was carried out using three washes of 2% ACN followed by three washes of 2% ACN containing 0.1% formic acid (Sigma). Elution of desalted peptides into glass capillary tubes was carried out using three rounds of 100 µL 60% ACN, 0.1% formic acid. Peptides were dried in a vacuum and resuspended in 2% ACN, 0.1% formic acid at 250 ng/µL. LC-MS/MS analyses: Liquid chromatography separation was done with a home-packed 100 µM ID X 20 mm precolumn connected to a 75 µM X 500 mm analytical column packed with C18 Luna beads (5µm diameter, 100Å pore size; Phenomenex 04A-5452). The columns were connected to an Ultimate 3500 RS nano UPLC system (Dionex). 1 µg of desalted peptides were loaded onto the column with starting mobile phase of 2% ACN, 0.1% formic acid. Peptides were eluted with the following gradient of 10 mins at 2% ACN, increase to 25% ACN for 115 mins, to 35 % ACN over 5 mins, to 80% ACN over 5 mins, followed by a column wash of 85% for 20 mins. The flow rate was constant at 300 µL/min. Typical backpressure values during separation were 5. The MS/MS spectra were acquired at a resolution of 17,500, with a target value of 2 × 105 ions or a maximum integration time of 120 ms and the isolation window was set at 4.0 m/z. Bioinformatics analyses: Maxquant version 1.3.0.5 was used to process the raw data for peptide and protein identification.19 Andromeda search engine used E. coli (K12 strain) reference proteome (Uniprot: UP000000625) which has 4305 protein groups as the targetdecoy database. MS/MS database search was performed using default settings, with a 20 ppm mass tolerance for the main search. Cysteine carbamidomethylation was selected as a fixed modification. Trypsin/P was selected as the protease, with up to three missed cleavages allowed. Results were filtered by a 0.01 false discovery rate at both protein and peptide levels. The minimum length of acceptable identified peptides was set as seven amino acids. The variability of the number of identified peptides and protein groups was calculated as the standard deviation of the corresponding numbers amongst triplicates. The physicochemical parameters of identified proteins/peptides, including the isoelectric point (pI), Kyle & Doolittle hydrophobicity (Hy) and molecular weight (MW) were calculated using the Protein Properties Analyses Software (ProPAS).

20

In silico digestion of

the E. coli-K12 reference proteome was performed using Protein Digestion Simulator V2.25350.

21

Parameters for the in silico digest included maximum and minimum fragment

mass set at 300 and 6000, respectively. In addition, the minimum residue count option was set at 7 and the ‘no trypsin mis-cleavages’ option was chosen. Phobius webserver was used for the prediction of transmembrane helices.22 PSORTdb 2.0 webserver was used for the determination of protein subcellular localisation.23,24 Identified protein locations with a 9 ACS Paragon Plus Environment


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threshold scoring of more than 7 were counted. Biological sequence alignment editor (BioEdit) was used for alignment and parsing of peptide and protein sequences.25

Results & Discussion For comparison between eFASP and FASP methods, the preparation of the proteomic material and the subsequent desalting of eluted peptides before LC-MS analyses were standardised to maintain consistency. Briefly, total protein was extracted from cell pellets harvested from a single E. coli K12 culture, lysed in RIPA buffer and reduced in the presence of 100 mM DTT final concentration. Although RIPA buffer contained 0.5% DCA, the buffer exchanges used in FASP would dilute the reagent prior to trypsin digestion, whereas DCA is maintained at a final concentration of 0.2% throughout eFASP buffer exchanges. In addition, the desalting of eluted peptides prior to LC-MS/MS analyses was carried out using C18 stage tips in place of phase transfer using ethyl acetate;4,18 we were concerned that the latter method was unsuitable for reproducible high throughput sample processing since it requires careful removal of peptide enriched aqueous layer from beneath the organic layer. The C18 stage tip desalting method was modified to circumvent precipitation of acid-insoluble DCA on column. Peptide rich solutions were loaded on C18 stage tips from either FASP or eFASP methods and were initially washed with 2% ACN solution followed by further washes with acidified 2% ACN containing 0.1% formic acid. Precipitation of DCA was observed after eFASP-derived peptides were desalted using the acidified wash step. To account for technical variance, both protein sample preparation methods were carried out in triplicate on the prepared cell lysate. Physicochemical properties of peptides


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An average of 13864 ± 667 and 11690 ± 3253 peptides were identified from LC-MS/MS analyses of each FASP and eFASP triplicate, respectively (Table 1). FASP gave a combined total of 14099 non-redundant peptides (NRPs), where 85.6 ± 1.2% of these were completely digested by trypsin. By comparison, eFASP gave 13414 NRPs and 83.3 ± 2.1 % of these were fully digested. Both methods share proportionally similar number of NRPs that contain either one (13.7 ± 1.1% and 15.7 ± 1.9%, respectively) or two (0.7 ± 0.1 % and 0.9 ± 0.2%, respectively) missed trypsin recognition sites (Table 1). Contrary to previous reports, our data thus does not support the conclusion that the inclusion of 0.2% DCA in the eFASP digestion buffer enhances trypsin activity. 4–11 Both methods shared 10196 identical NRPs and both had more than 3000 NRPs outliers that are probably unique to each method (Table 1). To determine whether these outlier NRPs are selectively enriched by either method, the physicochemical properties of the peptides were characterised. Scatter plots (Figure 1A-C) show significant overlap between most of these outliers and the shared NRPs, which suggests that these outliers do not in general arise as a result of their physicochemical properties. It is therefore possible that these overlapping outliers are instead due to technical variance amongst sample preparations and/or LC-MS/MS runs. To circumvent this, additional LC-MS/MS runs on the same sample and additional sample preparations replicates could be used to reduce technical variance.26 Notwithstanding the above, the scatter plots do show substantial differences in hydrophobicity and pI for those outlier NRPs that do not overlap with shared NRPs. To further characterise the differences between FASP and eFASP methods, box and whisker plots were used to provide a statistical comparison of these methods (Figure 2). In addition, peptides obtained from a silico digestion of Uniprot reference E. coli (strain K12) proteome was used for comparison of physicochemical properties. As expected the MW of NRPs are relatively constant across all sample preparations, with the majority found between mass 11 ACS Paragon Plus Environment


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ranges of 1079±29 and 1794±39 Da (Figure 2A). The MW of peptides is undoubtedly influenced by the extent of trypsin digestion and MWCO filtration unit used. The permeability of the filters may have reduced yields of peptides larger than 1800 Da, since there is approximately 20% difference amongst the upper quartiles of sample preparations compared with in silico peptides (Figure 2A). The upper quartile limit for in silico peptides is 2180 Da. According to Figure 1A and C, the FASP method purifies more unique hydrophilic NRPs within hydropathicity scale ranges of -1 to -3. This agrees with the box and whisker comparison of FASP outliers with that of eFASP outliers and shared peptides in Figure 2B. However, comparison of the majority (upper and lower quartiles) of all NRPs of FASP, eFASP and in silico tryptic digests show that they share similar hydropathicity scales, with small variations between the ranges of -0.476±0.082 and 0.472±0.047 (Figure 2B). A noticeable difference was observed between the pI ranges of the outliers NRPs of FASP and eFASP, with the majority of outlier eFASP NRPs lying above pI 7.5 whilst the majority of outlier FASP NRPs lie below 5.5 (Figure 2C). The majority of in silico peptides have a pI in the range 4.37 and 6.74 (Figure 2C). All FASP NRPs have a slightly smaller range of 4.25 and 6.04 whereas all eFASP NRPs have a broader range spanning from 4.46 to 8.22. eFASP seems therefore to enhance purification of more basic NRPs. Protein groups Assembly of peptides yielded 1486±17 and 1442± 125 protein groups for each sample preparation from FASP and eFASP, respectively (Table 1 and supplementary Table 1). The average peptide to protein coverage is 23.56% for FASP and 19.48% for eFASP. FASP and eFASP gave a total of 1723 and 1793 non-redundant protein groups (NRPGs), respectively. In addition these totals include 169 and 210 NRPGs, respectively that only have


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one peptide match (Table 1). The purification of higher pI NRPs in the case of eFASP possibly contributes to a marginally higher NRPGs total than FASP, despite its lower NRPs total. FASP and eFASP share 1581 NRPGs and both methods have 142 and 212 protein group outliers respectively (Table 1). There was no observable difference in the physicochemical properties of protein groups between the two outlier groups (data not shown). Although it has been reported that “in gel” proteolysis is a better choice for analyses of membrane proteins than “in solution” and “on filter” proteolysis, the eFASP method was reported to enhance recovery these proteins.4,27 To aid the comparison between proteins identified by the FASP and eFASP methods, the Phobius webserver was used to scan NRPGs obtained from both methods, as well as from the reference E. coli- K12 strain proteome, for transmembrane helices (TMH). There are 5722 predicted TMH derived from the 4305 protein groups of the E. coli- K12 reference proteome, equating to a ratio of 1.33 TMH per protein group. However, no difference was found between FASP and eFASP protocols, with each giving a recovery ratio of 0.73 TMH per protein group. A recent report - although not an FASP method - showed a substantial improvement in membrane protein recovery from cells where 5% DCA was used in place of SDS in the cell lysis/extraction step and 1% DCA was included in the digestion step.5 It is possible therefore that since SDS was used here at 1% final concentration in RIPA buffer, it bound tightly to highly hydrophobic peptides/proteins, thus affecting their purification.28 Finally, we sought to determine whether protein groups from specific cellular locations were preferentially recovered by either method. The Psort webserver was used to predict protein localisation, using the NRPGs of the FASP and eFASP protein groups and the E. coli- K12 reference proteome as inputs (Figure 3). Of 4305 protein groups in the E. coli- K12 reference proteome, the cellular location of 3372 protein groups is known. Our data shows that FASP 13 ACS Paragon Plus Environment


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and eFASP each extracted an average of ~55% of proteins associated with the cytoplasm and an average of 23% of extracellular proteins. Regarding membrane proteins, an average 47% of proteins from the outer membrane were extracted, whereas less than 30% of cytoplasmic membrane proteins were extracted. This lower recovery of membrane proteins presumably accounts for the low TMH to protein group ratio calculated for the FASP and eFASP NRPGs. Notably however, FASP extracted all known periplasmic associated proteins where eFASP only obtained 54%, possibly due to the lower pI range of NRPs associated with FASP meaning that periplasmic proteins are recovered more easily. Conclusion This technical note compares FASP with a recently reported enhanced FASP method using an uHPLC-Q Exactive system for analysis. The higher number of peptides and protein groups identified here compared to the original eFASP report enabled a more robust analysis of any perceived advantage of either method. Although the two methods showed a difference at the physicochemical pI of the peptides, there were only small, insignificant differences in the number of protein groups identified. Furthermore, although there have been previous reports of DCA improving membrane protein recovery using non-filter based sample preparation methods,5 our study did not find the same to be true for the FASP method. However, in the future it may be interesting to evaluate FASP protocols carried out on cell lysates that are prepared in 5% DCA instead of SDS. It must be stressed that FASP method is a useful method for the removal of chaotropes (in particular SDS) and detergents that were used to aid protein extraction and solubilisation in the precise protocols used here. By employing state-of-the-art LC-MS/MS shot gun proteomics, our results indicate that FASP and eFASP showed no significant differences at the protein identifications level when higher peptide and protein coverage is obtained. However, due to the slight differences in


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selectivity at the physicochemical level of identified peptides, these methods can be seen to be complementary for analyses of complex peptide mixtures. Associated context Supporting Information Available: Microsoft Excel spreadsheet containing protein groups obtained from Maxquant analyses of FASP and eFASP triplicates.

First Excel sheet

“ProteinGroups” is the raw data output. The second sheet “Filtered Rev and Cont” is where the reverse hits and contaminants sequences have been removed. The last sheet “FASP and eFASP” contains the relevant data that was used for the comparison of FASP and eFASP protein groups. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments We thank the National Research Foundation (NRF), South Africa, for funding. JMB thanks the NRF for a South African Research Chair Initiative grant. SG thanks the CSIR, South Africa, for a PhD bursary.



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TABLE LEGENDS Table 1. Summary of MS spectra, trypsin digestion efficiency, peptide and protein groups identified for each triplicate sample that were prepared using either FASP or eFASP methods. In addition, a summary of shared and outlier NRPs and NRPGs including identified transmembrane helices is included.

FIGURE LEGENDS Figure 1. Scatterplots of FASP and eFASP shared and outlier peptides based on their physicochemical properties. (A) Molecular weight (MW) versus hydropathicity (Hy), (B) Molecular weight versus isoelectric point (pI) and (C) Hydropathicity versus isoelectric point. Blue dots represent FASP outliers, orange eFASP outliers and black open circles are shared peptides between the two methods. Hydropathicity is based on Kyle and Doolittle’s (1982) grand average of hydropathicity index (GRAVY) scoring matrix.

Figure 2. Box and whiskers plots of physicochemical properties of peptides extracted using FASP & eFASP, including those that are shared between the two methods and the outliers as well as those that were generated from an in silico digest of E. coli (K12 strain) reference proteome. (A) Molecular weight (MW), (B) hydropathicity (Hy) and (C) isolectric point (pI). Hydropathicity is based on Kyle and Doolittle’s (1982) grand average of hydropathicity index (GRAVY) scoring matrix.

Figure 3. Predicted location of cellular proteins that were identified from FASP and eFASP extraction methods including known proteins from E. coli (strain K12) reference proteome.


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REFERENCES

(1)

Manza, L. L.; Stamer, S. L.; Ham, A.-J. L.; Codreanu, S. G.; Liebler, D. C. Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 2005, 5, 1742–1745.

(2)

Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 2009, 6, 359–362.

(3)

Wiśniewski, J. R.; Zielinska, D. F.; Mann, M. Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method. Anal. Biochem. 2011, 410, 307–309.

(4)

Erde, J.; Loo, R. R. O.; Loo, J. A. Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J. Proteome Res. 2014, 13, 1885–1895.

(5)

Lin, Y.; Lin, H.; Liu, Z.; Wang, K.; Yan, Y. Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics†. J. Sep. Sci. 2014, 37, 3321–3329.

(6)

Zhou, J.; Zhou, T.; Cao, R.; Liu, Z.; Shen, J.; Chen, P.; Wang, X.; Liang, S. Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane. J. Proteome Res. 2006, 5, 2547–2553.

(7)

Lin, Y.; Zhou, J.; Bi, D.; Chen, P.; Wang, X.; Liang, S. Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins. Anal. Biochem. 2008, 377, 259–266.

(8)

Masuda, T.; Sugiyama, N.; Tomita, M.; Ishihama, Y. Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines. Anal. Chem. 2011, 83, 7698– 7703.

(9)

Proc, J. L.; Kuzyk, M. A.; Hardie, D. B.; Yang, J.; Smith, D. S.; Jackson, A. M.; Parker, C. E.; Borchers, C. H. A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J. Proteome Res. 2010, 9, 5422–5437.

(10)

Masuda, T.; Tomita, M.; Ishihama, Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 2008, 7, 731–740.

(11)

Masuda, T.; Saito, N.; Tomita, M.; Ishihama, Y. Unbiased quantitation of Escherichia coli membrane proteome using phase transfer surfactants. Mol. Cell. Proteomics 2009, 8, 2770–2777.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

(12)

Tanca, A.; Biosa, G.; Pagnozzi, D.; Addis, M. F.; Uzzau, S. Comparison of detergentbased sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome. Proteomics 2013, 13, 2597–2607.

(13)

Sharma, R.; Dill, B. D.; Chourey, K.; Shah, M.; VerBerkmoes, N. C.; Hettich, R. L. Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization. J. Proteome Res. 2012, 11, 6008–6018.

(14)

Wiśniewski, J. R.; Rakus, D. Multi-enzyme digestion FASP and the “Total Protein Approach”-based absolute quantification of the Escherichia coli proteome. J. Proteomics 2014, 109, 322–331.

(15)

Michalski, A.; Damoc, E.; Hauschild, J.-P.; Lange, O.; Wieghaus, A.; Makarov, A.; Nagaraj, N.; Cox, J.; Mann, M.; Horning, S. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteomics 2011, 10, M111.011015.

(16)

Nagaraj, N.; Kulak, N. A.; Cox, J.; Neuhauser, N.; Mayr, K.; Hoerning, O.; Vorm, O.; Mann, M. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Mol. Cell. Proteomics 2012, 11, M111.013722.

(17)

Kelstrup, C. D.; Young, C.; Lavallee, R.; Nielsen, M. L.; Olsen, J. V. Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. J. Proteome Res. 2012, 11, 3487–3497.

(18)

Rappsilber, J.; Ishihama, Y.; Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 2003, 75, 663–670.

(19)

Cox, J.; Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26, 1367–1372.

(20)

Wu, S.; Zhu, Y. ProPAS: standalone software to analyze protein properties. Bioinformation 2012, 8, 167–169.

(21)

Monroe, M. Protien Digestion Simulator http://omics.pnl.gov/software/proteindigestion-simulator (accessed Nov 11, 2014).

(22)

Käll, L.; Krogh, A.; Sonnhammer, E. L. L. Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server. Nucleic Acids Res. 2007, 35, W429–W432.

(23)

Rey, S.; Acab, M.; Gardy, J. L.; Laird, M. R.; deFays, K.; Lambert, C.; Brinkman, F. S. L. PSORTdb: a protein subcellular localization database for bacteria. Nucleic Acids Res. 2005, 33, D164–D168.


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24)

Yu, N. Y.; Laird, M. R.; Spencer, C.; Brinkman, F. S. L. PSORTdb--an expanded, auto-updated, user-friendly protein subcellular localization database for Bacteria and Archaea. Nucleic Acids Res. 2011, 39, D241–D244.

(25)

Hall, T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids Symp Ser 1999, 41, 95–98.

(26)

Ham, B. M.; Yang, F.; Jayachandran, H.; Jaitly, N.; Monroe, M. E.; Gritsenko, M. A.; Livesay, E. A.; Zhao, R.; Purvine, S. O.; Orton, D.; et al. The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage. J. Proteome Res. 2008, 7, 2215–2221.

(27)

Choksawangkarn, W.; Edwards, N.; Wang, Y.; Gutierrez, P.; Fenselau, C. Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins. J. Proteome Res. 2012, 11, 3030–3034.

(28)

Liebler, D. C.; Ham, A.-J. L. Spin filter-based sample preparation for shotgun proteomics. Nat. Methods 2009, 6, 785; author reply 785–786.



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A

B

C

Figure 1. Scatterplots of FASP and eFASP shared and outlier peptides based on their physicochemical properties. (A) Molecular weight (MW) versus hydropathicity (Hy), (B) Molecular weight versus isoelectric point (pI) and (C) Hydropathicity versus isoelectric point (pI). Blue dots represent FASP outliers, orange eFASP outliers and black open circles are shared peptides between the two methods. Hydropathicity is based on Kyle and Doolittle’s (1982)

Grand Average of Hydropathicity index (GRAVY) scoring matrix.

ACS Paragon Plus Environment


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A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B

C

Figure

2.

Box

and

whiskers

plots

of

physicochemical properties of peptides extracted using FASP & eFASP, including those that are shared between the two methods and the outliers as well as those that were generated from an in silico digest of Escherichia coli (K12 strain) reference proteome. (A) Molecular weight (MW), (B) hydropathicity (Hy) and (C) isolectric point (pI). Hydropathicity is based on Kyle and Doolittle’s (1982) grand average of hydropathicity index (GRAVY) scoring matrix. The following symbols represent the following: (■) outlier, (×) median, (┴) upper quartile maximum, (┬) lower quartile

minimum. ACS Paragon Plus Environment

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Figure 3. Predicted location of cellular proteins that were identified from FASP and eFASP extraction methods including known proteins from E. coli

(strain K12) reference proteome.



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Comparative Reevaluation of FASP and Enhanced FASP Methods by

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