Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2‐D peptide separations employing mixed mode reversed phase anion exchange (MM (RP‐AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP‐AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP‐AX) compared with the traditional SCX hyphenated to RP‐LC‐MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP‐AX) for 2‐D LC proteomic studies was also extended in the analysis of iTRAQ‐labelled HeLa and cyanobacterial proteomes using nano‐flow chromatography interfaced to the MS/MS. We demonstrate MM (RP‐AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.     

High pH reversed‐phase chromatography as a superior fractionation scheme compared to off‐gel isoelectric focusing for complex proteome analysis

MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off‐gel IEF (OG‐IEF) and high pH RP (Hp‐RP) column chromatography have both been successfully utilized as a first‐dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12‐fraction replicate analysis, Hp‐RP resulted in more peptides and proteins identified than OG‐IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp‐RP. This leads to a more uniform distribution of total and unique peptides for Hp‐RP across all fractions collected. These results suggest that fractionation by Hp‐RP over OG‐IEF is the better choice for typical complex proteome analysis.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.     

Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy

In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies.     

Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.     

Gel‐free mass spectrometry analysis of Drosophila melanogaster heads

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under‐represented group in proteomics datasets. Herein, a bottom‐up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem‐mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter‐aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed‐phase (RP) LC at high pH in the first dimension and acidic RP‐LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 ( http://proteomecentral.proteomexchange.org/dataset/PXD001712 ).     

Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.     

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.     

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.     

Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure--beginning with isolated membrane fraction and finishing with MS data acquisition--takes 4-5 d.     

Identification of cross‐reactive B‐cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI‐TOF MS

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross‐allergenicity described between soy and milk proteins. We have previously identified several cross‐reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1‐casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α‐casein‐specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross‐reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI‐TOF MS analysis. On a second approach, the peptide mixture was resolved by RP‐HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI‐TOF MS. This novel MS based approach led us to identify and characterize four peptides on α‐casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross‐reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross‐reactivity, to further develop new and more effective vaccines for food allergy.     

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.     

Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus strong cation exchange (SCX) for fractionation of iTRAQ-labeled peptides

The iTRAQ technique is popular for the comparative analysis of proteins in different complex samples. To increase the dynamic range and sensitivity of peptide identification in shotgun proteomics, SCX chromatography is generally used for the fractionation of iTRAQ-labeled peptides before LC-MS/MS analysis. However, SCX suffers from clustering of similarly charged peptides and the need to desalt fractions. In this report, SCX is compared with the alternative ERLIC method for fractionating iTRAQ-labeled peptides. The simultaneous effect of electrostatic repulsion and hydrophilic interaction in ERLIC results in peptide elution in order of decreasing pI and GRAVY values (increasing polarity). Volatile solvents can be used. We applied ERLIC to iTRAQ-labeled peptides from rat liver tissue, and 2745 proteins and 30,016 unique peptides were identified with high confidence from three technical replicates. This was 12.9 and 49.4% higher, respectively, than was obtained using SCX. In addition, ERLIC is appreciably better at the identification of highly hydrophobic peptides. The results indicate that ERLIC is a more convenient and more effective alternative to SCX for the fractionation of iTRAQ-labeled peptides. Quantification data show that both SCX and ERLIC fractionation have no significant effect on protein quantification by iTRAQ.     

Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC-MS

A method for high-resolution proteomics analyses of complex protein mixtures is presented using multidimensional HPLC coupled to MS (MDLC-MS). The method was applied to identify proteins that are differentially expressed during fruit ripening of tomato. Protein extracts from red and green tomato fruits were digested by trypsin. The resulting highly complex peptide mixtures were separated by strong cation exchange chromatography (SCX), and subsequently analyzed by RP nano-LC coupled to quadrupole-TOF MS. For detailed quantitative comparison, triplicate RP-LC-MS runs were performed for each SCX fraction. The resulting data sets were analyzed using MetAlign software for noise and data reduction, multiple alignment and statistical variance analysis. For each RP-LC-MS chromatogram, up to 7000 mass components were detected. Peak intensity data were compared by multivariate and statistical analysis. This revealed a clear separation between the green and red tomato samples, and a clear separation of the different SCX fractions. MS/MS spectra were collected using the data-dependent acquisition mode from a selected set of differentially detected peptide masses, enabling the identification of proteins that were differentially expressed during ripening of tomato fruits. Our approach is a highly sensitive method to analyze proteins in complex mixtures without the need of isotope labeling.     

Comparison of sample preparation techniques for large‐scale proteomics

Numerous workflows exist for large‐scale bottom‐up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in‐solution digests, SDS‐PAGE coupled with in‐gel digestion, gel‐eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high‐/low‐pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS‐PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.     

Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome

Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.     

Degradation of milk‐based bioactive peptides by yogurt fermentation bacteria*

Aims: To analyse the effect of cell‐associated peptidases in yogurt starter culture strains Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST) on milk‐protein‐based antimicrobial and hypotensive peptides in order to determine their survival in yogurt‐type dairy foods. Methods and Results: The 11mer antimicrobial and 12mer hypotensive milk‐protein‐derived peptides were incubated with mid‐log cells of LB and ST, which are required for yogurt production. Incubations were performed at pH 4·5 and 7·0, and samples removed at various time points were analysed by reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The peptides remained mostly intact at pH 4·5 in the presence of ST strains and moderately digested by exposure to LB cells. Peptide loss occurred more rapidly and was more extensive after incubation at pH 7·0. Conclusions: The 11mer and 12mer bioactive peptides may be added at the end of the yogurt‐making process when the pH level has dropped to 4·5, limiting the overall extent of proteolysis. Significance and Impact of the Study: The results show the feasibility of using milk‐protein‐based antimicrobial and hypotensive peptides as food supplements to improve the health‐promoting qualities of liquid and semi‐solid dairy foods prepared by the yogurt fermentation process.     

Expanding the cerebrospinal fluid endopeptidome

Biomarkers of neurodegenerative disorders are needed to assist in diagnosis, to monitor disease progression and therapeutic interventions, and to provide insight into disease mechanisms. One route to identify such biomarkers is by proteomic and peptidomic analysis of cerebrospinal fluid (CSF). In the current study, we performed an in‐depth analysis of the human CSF endopeptidome to establish an inventory that may serve as a basis for future targeted biomarker studies. High‐pH RP HPLC was employed for off‐line sample prefractionation followed by low‐pH nano‐LC‐MS analysis. Different software programs and scoring algorithms for peptide identification were employed and compared. A total of 18 031 endogenous peptides were identified at a FDR of 1%, increasing the number of known endogenous CSF peptides 10‐fold compared to previous studies. The peptides were derived from 2 053 proteins of which more than 60 have been linked to neurodegeneration. Notably, among the findings were six peptides derived from microtubule‐associated protein tau, three of which span the diagnostically interesting threonine‐181 (Tau‐F isoform). Also, 213 peptides from amyloid precursor protein were identified, 58 of which were partially or completely within the sequence of amyloid β 1–40/42, as well as 109 peptides from apolipoprotein E, spanning sequences that discriminate between the E2/E3/E4 isoforms of the protein.     

Membrane protein shaving with thermolysin can be used to evaluate topology predictors

Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large‐scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over‐expressed proteins. Second, we analyzed the peptides from non‐over‐expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.