Novel Highly Sensitive, Specific, and Straightforward Strategy for

Communication pubs.acs.org/jpr

Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N‑Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase Icp55 A. Saskia Venne,† F.-Nora Vögtle,‡ Chris Meisinger,‡,§ Albert Sickmann,†,∥ and René P. Zahedi*,† †

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany Institut für Biochemie und Molekularbiologie, ZBMZ and §BIOSS (Centre For Biological Signalling Studies), Universität Freiburg, Germany ∥ Medizinisches Proteom Center, Bochum, Germany ‡

S Supporting Information *

ABSTRACT: We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC−MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 μg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities. KEYWORDS: degradomics, N-terminal, COFRADIC, Icp55, protease, peptidase

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INTRODUCTION Proteolysis is one of most important post-translational modifications and is involved in cellular signaling,1 protein translocation,2−4 and protein degradation. It plays an important role in vital processes such as hemostasis or inflammation5 and thus is a key player in cell life and death. Proteolytic cleavage leads to the generation of novel protein N-termini; consequently, methods for enrichment, identification, and differential quantitation of N-terminal peptides are of utmost interest for protease and peptidase research. Already in 1962 Edman degradation6 was used to identify the N-terminal amino acids of human haptoglobins.7 With the advent of massspectrometry-based proteomics, innovative techniques such as COFRADIC8 and TAILS9 were developed. These methods represented a major step forward in protease research because they allowed, for the first time, to specifically identify protein N-termini and neo-N-termini from highly complex samples such as tissues, cells, and organelles due to the specific enrichment of N-terminal peptides from the vast excess of internal peptides typically present in proteolytic digests.10−12 Here we present a novel technique for enriching N-terminal peptides based on a 2D, charge-based SCX separation,13 which © 2013 American Chemical Society

we term charge-based fractional diagonal chromatography (ChaFRADIC). It is straightforward to implement, does not require elaborate chemistry, has a high specificity, and can be utilized to differentially quantify N-proteomes with a good coverage starting from merely a few micrograms of cell lysate per condition. The ChaFRADIC workflow comprises a total of five major steps (see Figure 1): (1) Proteins are carbamidomethylated and primary amines (Lys residues and free protein N-termini) are dimethylated,14 allowing the incorporation of stable isotope labels for differential analyses. (2) Thus, two (or three) differentially labeled samples can be pooled and subsequently subjected to tryptic digestion. Here trypsin cleaves which ArgC specificity, as Lys residues are blocked due to dimethylation. Upon digestion, two major classes of peptides are generated: N-terminal peptides with blocked (dimethylated) N-termini and internal peptides with free N-termini. (3) The peptide mixture is then separated by optimized strong cation exchange chromatography (SCX) at pH 2.7, which allows us to selectively collect charge states Received: May 7, 2013 Published: August 7, 2013 3823

dx.doi.org/10.1021/pr400435d | J. Proteome Res. 2013, 12, 3823−3830

Journal of Proteome Research

Communication

Figure 1. Scheme of the ChaFRADIC workflow. (1) Disulfide bonds are reduced, free cysteines are carbamidomethylated, and Lys residues as well as free protein N-termini are dimethylated on the protein level. (2) Samples are pooled and digested with trypsin (ArgC specificity due to the dimethyl labeling of Lys residues), generating mixtures of N-terminal and internal peptides with different charge states at pH 2.7. (3) The pooled sample is separated using an optimized SCX gradient and charge states are selectively collected. (4) Free N-termini of internal peptides are deuteroacetylated for each charge -state fraction separately, inducing a charge-state reduction, whereas N-terminal peptide charge states remain unchanged. (5) Each derivatized sample is subjected to a second SCX separation under the same conditions, and only those peptides retaining their charge states are collected for the subsequent LC−MS analysis. In total, the following fractions are collected: +1 → +1, +2 → +2, +3 → +3, +4 → +4, and +>4 → +>4. The entire workflow from 1−5 including subsequent LC−MS analysis can be conducted in 3 to maximum 4 days. FT: flow through.

+1, +2, +3, +4, and >+4. (4) Next, each of the five charge fractions is treated with NHS trideutero-acetate. Thus, free N-termini of internal peptides are acetylated, whereas blocked N-terminal peptides remain unchanged. This step leads to a selective reduction of internal peptide charge states at pH 2.7.

The trideutero-acetyl group allows us to clearly distinguish derivatized internal peptides from endogenously acetylated N-terminal peptides. (5) Each of the five derivatized chargestate fractions is then separated by SCX using the same conditions as before. Because of the acetylation step, internal 3824



Communication

Dimethyl Labeling on the Protein Level

peptides will elute within earlier retention time/charge state windows, whereas retention time windows of N-terminal peptides remain unchanged, allowing the selective enrichment of the latter class. Using a 1:1 mixture of 50 μg of S. cerevisiae lysate each, we demonstrate that the workflow is reproducible and specific (78% of the identified nonredundant peptides are N-terminal) and can be utilized for quantitation. A total of 1754 nonredundant N-terminal peptides could be identified and 1459 nonredundant N-terminal peptides could be quantified. To our knowledge, this represents so far the fastest and most sensitive method for N-terminal proteomics, even when compared with the recently developed PTAG (phospho tagging) method by Mommen et al.15 We further evaluated the performance of ChaFRADIC by differentially comparing mitochondria isolated from wild-type and icp55Δ yeast. As we previously demonstrated, Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule,16 thus stabilizing the respective share of the mitochondrial proteome. Using ChaFRADIC, only 30 μg of starting material per condition, and 10 h of LC−MS/MS time, we could quantify 732 N-terminal peptides from 396 proteins and identified 14 novel substrates of Icp55, not identified in our previous study, which indeed required about one hundred times more starting material, the usage of three different proteases (trypsin, chymotrypsin, V8), and the acquisition of more than 100 LC−MS analyses.

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For specific dimethylation of free N-termini and lysine residues, samples were incubated in either “light” or “heavy” labeling buffer for 2 h at 37 °C according to Jentoft et al.19 For dimethyl labeling, the light buffer comprised 20 mM CH2O, 40 mM NaBH3CN, and 200 mM HEPES, adjusted to pH 8.0 before addition to the sample. The corresponding heavy buffer comprised 20 mM CD2O, 40 mM NaBD3CN, and 200 mM HEPES, pH 8.0. Excess formaldehyde was quenched by the addition of glycine to a final concentration of 60 mM for 10 min. Afterward, samples were treated with 130 mM hydroxylamine for 15 min at room temperature. Finally, the differentially labeled samples were pooled 1:1 for further sample preparation. Proteolytic Digestion

Prior to proteolytic digestion the pooled samples were subjected to ethanol precipitation. In brief, samples were diluted 10-fold with ice-cold ethanol and stored at −40 °C for 1 h, followed by centrifugation at 4 °C at 16 000g for 30 min. The supernatant was carefully removed and protein pellets were resolubilized in 2 M GuHCl, 50 mM Na2HPO4, pH 7.8. The protein solution was further diluted 10-fold with 50 mM NH4HCO3, 5% ACN, and 1 mM CaCl2. For proteolytic digestion, trypsin was added in a 1:20 (w/w) ratio and samples were incubated for 15 h at 37 °C. Digest efficiency was controlled as described previously,20 and generated peptides were desalted using C18 solid-phase tips (SPEC C18 AR, 4 mg bed, Agilent Technologies, Böblingen, Germany) according to the manufacturer’s instructions. Eluted peptides were dried to completeness under vacuum, followed by resolubilization in 52 μL of SCX buffer A (10 mM KH2PO4, 20% ACN, pH 2.7). To control label efficiencies, we measured an aliquot corresponding to 0.5 μg of peptide by a 2 h LC−MS/MS analysis on an LTQ Orbitrap Velos (Thermo Scientific), as described previously.21 Labeling efficiency was determined by comparing the total number of lysines in all identified peptide− spectrum matches (PSMs) to the number of labeled lysines at 1% FDR and was typically above 95%.

MATERIALS

Materials and Reagents

Guanidine hydrochloride (GuHCl), hydroxylamine, sodium cyanoborodeuteride (NaBD3CN, 96 atom % D), Iodoacetamide (IAA), glycine, T-1426 trypsin, calcium chloride (CaCl2) and formaldehyde-D2 solution (CD2O, ∼20 wt % in D2O, 98 atom % D) were purchased from Sigma Aldrich (Steinheim, Germany). Formaldehyde (CH2O) and 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) were obtained from Applichem (Darmstadt, Germany), 1,4-dithio-DL-threithol (DTT) from Roche (Mannheim, Germany). All other chemicals were acquired from Merck (Darmstadt, Germany), and all ULC/MS-grade solvents were obtained from Biosolve (Valkenswaard, The Netherlands). NHS trideutero-acetate was synthesized as described previously.17

Enrichment of N-Terminal Peptides Using ChaFRADIC

SCX separations were performed using a U3000 HPLC system (Thermo Scientific) and a 150 × 1 mm POLYSULFOETHYL A column (PolyLC, Columbia, US, 5 μm particle size, 200 Å pore size) in combination with a tertiary buffer system consisting of SCX buffer A (10 mM KH2PO4, 20% ACN, pH 2.7), SCX buffer B (10 mM KH2PO4, 188 mM KCl, 20% ACN, pH 2.7), and SCX buffer C (10 mM KH2PO4, 800 mM NaCl, 20% ACN, pH 2.7). 50 μL of the resolubilized peptides was separated at a flow rate of 80 μL/min. Peptides were separated with an optimized gradient to efficiently separate different charge states, and fractions were automatically collected using the U3000 fractionation option. The gradient was as follows: 100% A for 10 min, followed by a linear increase from 0 to 20% B in 18 min. Afterward, B was kept at 20% for 10 min, followed by a linear increase from 20% to 40% B in 2 min. Then, B was kept at 40% for 5 min and linearly increased to 100% in 5 min. After 5 min at 100% B, C was set to 100% for 5 min. In each first-dimension SCX separation, five fractions corresponding to the charge states +1, +2, +3, +4, and >+4 were collected. Collected fractions were reduced to ∼40 μL under vacuum and brought to 300 μL with 200 mM Na2HPO4, pH 8.0 to a final pH of ∼7.0. Next, free N-termini of internal peptides were derivatized with NHS-trideutero acetate in two steps, modified

Sample Preparation

S. cerevisiae spheroblasts were prepared as described previously,18 icp55Δ and wild-type mitochondria were isolated as described by Vögtle et al.16 Samples were lysed in 6 M GuHCl, 50 mM Na2HPO4, pH 7.8 on ice, followed by heating at 85 °C for 5 min. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Thermo Scientific). Proteins were reduced with 10 mM DTT for 30 min at 56 °C and subsequently carbamidomethylated by incubation with 20 mM IAA for 30 min at room temperature in the dark. The yeast sample was split in two aliquots, and 50 μg each were used for heavy and light dimethyl labeling, respectively. For icp55Δ and wild-type mitochondria the starting amount after lysis was 30 μg of protein each. 3825



Communication

from Staes et al.22 Initially, NHS-trideutero acetate was added to a final concentration of 20 mM and samples were incubated at 37 °C for 1 h, followed by the addition of 10 mM NHStrideutero acetate under the same reaction conditions. After 2 h of total incubation time the reaction was quenched using 60 mM glycine and 130 mM hydroxylamine, and peptides were desalted as described above. Subsequently, dried peptides were resolubilized in 50 μL of SCX buffer A, separated and fractionated in the second SCX dimension under exactly the same conditions as mentioned above. Collected fractions were dried, resolubilized in 0.1% TFA, and desalted using C18 Supel 10 μL pipette tips (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instructions.

confidence corresponding to an FDR < 1% on the PSM level and search engine rank 1 were used. To compensate for deuterium-induced retention time shifts, the maximum window for corresponding peptides was set to 1 min. To generate the final list of N-terminal peptides (Supplemental Table 1 in the Supporting Information), only sequences of at least six amino acids, representing the first 100 amino acids of the respective protein’s sequence, were considered. To generate the final nonredundant protein N-termini list, we considered only unique peptides, and only the first six amino acids of all peptides were compared to remove duplicates derived from missed cleavages etc.; icp55Δ and wild-type mitochondria data were interpreted as previously described.16

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nano-LC−MS/MS

RESULTS AND DISCUSSION A most important step in quantitative proteomics is to evaluate the performance and reproducibility of a workflow and therefore assess its usability for robust quantitation, particularly with regard to limited sample amounts. After establishing our novel method for N-terminal enrichment, we thus conducted a control experiment, in which we labeled 50 μg of yeast digest each with light and medium dimethyl, respectively. The initial LC−MS analysis of the global digested sample indicated a high labeling efficiency (96% of Lys residues and N-termini) and reproducibility with a median (non-normalized) ratio of 1.1, close to the ideal ratio of 1.0. Next, we evaluated the efficiency to enrich and quantify for N-terminal peptides using our ChaFRADIC method. Analyzing the five obtained SCX fractions by LC−MS resulted in the identification of 8438 N-terminal PSM (out of 10 918 total PSM, 77%), corresponding to 1775 nonredundant N-terminal peptides, of which 1480 could be quantified. 40 peptides with fold changes >2 between both samples were manually checked to compensate for outliers derived from isotope pattern interferences and deuteriuminduced retention time shifts, resulting in the exclusion of 21 peptides. The remaining 1459 quantified N-terminal peptides had a median ratio of 1.1. (See Figure 2.) When considering

For LC−MS analysis, the dried C18 eluates were resuspended in 15 μL of 0.1% TFA and analyzed by nano-LC−MS/MS using a Q-Exactive mass spectrometer (Thermo Scientific) online coupled to a nano RSLC HPLC system (Thermo Scientific). For yeast samples, depending on the UV intensities in the second-dimension SCX separation, either 1/3 (+3, +5) or 2/3 (all others) of the fractions (v/v) were analyzed. For mitochondria, only 1/3 (v/v) was used per fraction. Samples were loaded onto a trap column (C18, 100 μm × 2 cm PepMap RSLC, Thermo Scientific) at a flow rate of 20 μL/min 0.1% TFA and subsequently separated on a 50 cm main column (C18, 75 μm × 50 cm PepMap RSLC, Thermo Scientific) using a binary gradient consisting of solvent A (0.1% FA) and solvent B (0.1% FA, 84% ACN) at a flow rate of 250 nL/min and 60 °C. Gradients increased linearly from 3 to 42% B in either 90 min (mitochondria) or 127 min (yeast). The Q-Exactive was operated in data-dependent acquisition mode acquiring full MS Scan at R = 70,000, followed by MS/MS of the 15 most abundant ions (Top15) at R = 17,500. Target values and fill times were set to 3 × 106 and 120 ms for MS and 5 × 104 and 250 ms for MS/MS, respectively. Charge states +2 to +5 were selected for fragmentation with a normalized collision energy of 30, and 10% NH4OH solution was placed in front of the ion source for charge-state reduction, as described by Thingholm et al.23 For analyzing SCX charge-state +1 fractions, the fragmentation of singly charged peptides was allowed as well while excluding m/z values of singly charged ions occurring in previous blank runs. Data Interpretation

Raw data were searched against an SGD database (September 2011; 6717 target sequences) using Mascot 2.4 (Matrix Science) and the Proteome Discoverer software version 1.3 (Thermo Scientific) including the precursor ions quantifier and percolator24 nodes, employing a two-step strategy and semiArgC enzyme specificity with a maximum of two missed cleavage sites. First, dimethylation light (+28.0313 Da) and dimethylation medium (+34.0689 Da) at Lys and N-termini were set as variable and carbamidomethylation of Cys (+ 57.0214 Da) as fixed modifications. Second, N-terminal acetylation (+ 42.0105 Da) as well as dimethylation light/ medium at Lys were set as variable and carbamidomethylation of cysteines as fixed modification. The reason to employ this two-step search is the occurrence of endogenously acetylated N-termini, which required the usage of two different settings in the precursor ion quantifier node because it cannot handle labeled N-termini (light/medium dimethyl) in conjunction with nonlabeled N-termini (acetylation). Mass tolerances were set to 10 ppm for MS and 0.02 Da for MS/MS. As filters, high

Figure 2. Robust quantitation of yeast N-terminal peptides using ChaFRADIC. 100 μg of yeast proteins was split into two aliquots and subjected to the workflow depicted in Figure 1, employing light and medium dimethyl labeling. In 10 h of LC−MS, 1459 nonredundant N-terminal peptides were quantified from the 1:1 pooled yeast sample, yielding a median ratio of 1.1.

(i) only peptides that start within the first 100 amino acids of the respective protein’s sequence and (ii) only the most Nterminal peptide per protein, our approach led to the identification of 806 unique protein N-termini, representing >20% of the expected yeast proteome,25 with 661 unique 3826



Communication

existing data from large-scale studies. Indeed 60% of these 806 sequences were previously identified. Because most proteomic studies employ trypsin for digestion, whereas our study has an Arg-C specificity, we furthermore converted our 806 sequences into fully tryptic sequences. Thus, we obtained a list of 532 fully tryptic peptide sequences with at least six amino acids, out of which 75% were identified in previous studies within the GPM. This extensive overlap with previous data further demonstrates the validity of our approach. Finally, to assess the performance of our novel strategy for identifying differential N-terminal peptides for degradomics, we compared mitochondria isolated from wild-type and icp55Δ yeast. Icp55 is peptidase which is critical for protein stability in mitochondria, identified by us in the so-far most comprehensive analysis of mitochondrial N-proteome using N-terminal COFRADIC, which comprised consumption of more than 3 mg of protein, the acquisition of almost 100 LC−MS files, and the usage of three different proteases (trypsin, chymotrypsin, V8).16 Here we used only 30 μg of protein per condition to apply our ChaFRADIC workflow and analyzed the five obtained fractions enriched in N-terminal peptides. We quantified 627 nonredundant N-terminal peptides (median 1.0) and identified an additional 77 endogenously N-terminal acetylated peptides. Because on the one hand the isolation of mitochondria is a complex process and on the other hand Icp55 substrates should have clearly differential signals, we only considered those 114 peptides showing a greater than five-fold change between the two samples. Notably, for some Icp55 substrates both N-termini were quantified, the non-Icp55 processed N-terminus obtained after MPP cleavage (mainly in icp55Δ mitochondria) and the mature N-terminus after consecutive MPP/Icp55 cleavage (mainly in wild-type mitochondria). Thus, for Atp3 the N-terminal peptide 33 YATLKEVEMR42 was 7.2 times up-regulated in icp55Δ mitochondria, whereas its mature form 34ATLKEVEMR42 was more than 1000 times up-regulated in wild-type mitochondria. We screened the resulting list for potential Icp55 substrates, considering (i) subcellular localization of the protein, (ii) only those peptides which were the most N-terminal for the respective protein, and (iii) the presence of the established Icp55 consensus motifs R/K-X-L, R/K-X-F, and R/K-X-Y, respectively. We thus obtained a list of 36 nonredundant Icp55 substrates, of which 14 were unknown so far, as summarized in Table 1. Considering that using the applied strategy 9 of the 38 established Icp55 substrates are most unlikely to be detected, based on the length of the peptides generated with the applied ArgC digestion specificity, we covered 76% of the accessible Icp55 substrates (22 out of 29). Notably, these results were obtained with ∼1% of the starting material and within ∼1% of the time previously required.16 All of the 14 novel substrates identified in this study contain the classical MPP cleavage site motif, a positively charged Arg or Lys residue in position −2 of the first processing site. Following MPP cleavage, Icp55 removes Leu, Phe, or Tyr. The most interesting discovery, however, is the novel substrate Isa2. After processing by MPP this protein is likely processed in two consecutive steps, in which Icp55 removes two destabilizing amino acids: first Phe and then in a second round of processing Tyr, resulting in the mature stable protein. This would be the first example of a sequential trimming by Icp55 and of a triple processing event in a mitochondrial precursor protein.

protein N-termini quantified between the two samples. Notably, these results were obtained from only five LC−MS analyses of merely ∼40% of the enriched fractions and starting with only 100 μg of sample in total, clearly demonstrating the high reproducibility, robustness, and efficiency of the presented workflow for differential N-terminal proteomics. Such results can be obtained within three days for the complete procedure (see Figure 1), employing a total of only six SCX separations of 80 min each and a total 10 h of LC−MS analysis; indeed, because of the comparably low complexity of the obtained fractions, LC−MS gradients can be further shortened to 1 h. We compared our data to a recent study from Mommen et al. who employed a phospho-tagging (PTAG) approach for N-terminal enrichment. After removing redundant sequences, derived from the usage of three different enzymes, missed cleavage sites, and so on, and considering only the most N-terminal peptide per protein, Mommen et al. identified 547 protein N-termini compared with 806 (∼1.5-fold increase) identified and 661 quantified N-termini in our study. 229 N-termini overlap between both studies, whereas 318 are unique to Mommen et al. and 577 to our data set. (See Figure 3.) These

Figure 3. Summary of the N-proteome identified by ChaFRADIC. (A) All 806 identified N-termini were assigned to bins (1, 2, 3−10, 11−20, 21−30, ..., 91−100) representing the position of their starting amino acid within the respective protein’s sequence. (B) Comparison between yeast N-proteomes derived from ChaFRADIC (this study) and PTAG (Mommen et al.). For 35 of the 229 (∼15%) overlapping proteins the N-termini differed.

at-first-glance complementary specificities can be partially attributed to the application of different digestion strategies (merely trypsin in the case of ChaFRADIC compared with a combination of trypsin, V8, and chymotrypsin in the case of PTAG), also contributing to a slight shift toward shorter N-terminal peptide sequences in the case of ChaFRADIC (median of 13 ± 6 amino acids compared with 17 ± 6 for PTAG), whereas the number of Lys/Arg/His residues that can bear positive charges is similar (median of 2). Considering (i) the complexity of the workflows (chemistry, digestion conditions, amounts of starting material, and number of LC−MS analyses) and (ii) the expenditure of time, as well as the facts that we (iii) differentially quantified two samples and thus even doubled the complexity of the sample and (iv) did not remove N-terminal pyroglutamate,17 our method is more sensitive and straightforward compared with PTAG N-terminal enrichment. We searched our list of 806 N-terminal peptide sequences against the Global Proteome Machine (GPM, http://www.thegpm.org/)26 to determine the overlap with 3827


3828

FMP16

MRP20

RSM28

FMP10

TIM21

AIM17

COX6

ACP1

HSP60

ACO1

MSK1

ATP11

CIT1

IDH2

RDL2

MYS1

COX11

MSF1

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

YPR047W

YPL132W

YPL097W

YOR286W

YOR136W

YNR001C

YNL315C

YNL073W

YLR304C

YLR259C

YKL192C

YHR051W

YHL021C

YGR033C

YER182W

YDR494W

YDR405W

YDR070C

YDL202W

YDL178W

YDL130W-A

YBR039W

ORF

start

33

34

23

24

36

31

14

35

14

15

14

42

23

24

40

36

37

21

22

17

30

35

37

38

15

16

26

37

38

46

18

ICPΔ

↑↑

↓↓

↑↑

↓↓

↑↑

↑↑

↓↓

↓↓

↑↑

↓↓

↓↓

↑↑

↑↑

↓↓

↑↑

↑↑

↓↓

↑↑

↓↓

↓↓

↓↓

↑↑

↑↑

↓↓

↑↑

↓↓

↓↓

↑↑

↓↓

↓↓

↓↓ 51

63

52

52

39

25

25

48

48

44

45

24

34

34

48

48

55

44

34

58

28

29

29

52

19

40

43

43

43

42

42

end

Y

Y

Y

F

L

Y

Y

L

Y

Y

Y

Y

Y

L

L

Y

Y

removed AA

sTLkVPHVEINGIkYkTDPQTTNVTDSIIkLTDR

sVNSEQPkHTFDISkLTR

sQPSALEVQGTSDSR

ySQPSALEVQGTSDSR

tTkAPkIYTFDQVR

aTVkQPSIGR

1ATVkQPSIGR

sSASEQTLkER

ySSASEQTLkER

ySSSPEQkYR

sLAHAVDTSkMEATRR

aTVSNLTR

sSHkELkFGVEGR

ySSHkELkFGVEGR

sANLSkDQVSQR

ySANLSkDQVSQR

ySDAHDEETFEEFTAR

tSAATAAAANRGHIIkTYFNR

yTSAATAAAANR

ySNGTGATSGkkDDkTR

tNGVVFkTASkPkRR

sTSVTEDFINSILAR

ySTSVTEDFINSILAR

aSVVESSSkILDkSGSDR

mRkSPR

yALASEQPSR

ySTkIQTR

sDGPLGGAGPGNPQDIFIkR

ySDGPLGGAGPGNPQDIFIkR

aTLkEVEMR

yATLkEVEMR

identified sequence

36

35

34

33

32

31

30

29

28

27

26

25

24

23

no

SDH4

ATP12

ISA2

TAM41

MSD1

AEP2

YKL023C-A

P0S5

FCJ1

MRS2

COQ2

YHR056W-A

MTG2

COQ8

gene

YDR178W

YJL180C

YPR067W

YGR046W

YPL104W

YMR282C

YKL023C-A

YPL188W

YKR016W

YOR334W

YNR041C

YHR056W-A

YHR168W

YGL119W

ORF

↓↓

↓↓

↑↑

↓↓

↑↑

↑↑

↓↓

↑↑

↓↓

↓↓

↓↓

↑↑

↓↓

↑↑

↑↑

↓↓

↓↓

↑↑

↓↓

↑↑

ICPΔ

33

24

23

36

35

27

17

16

68

13

18

17

18

32

34

51

24

23

42

41

start

48

46

46

49

49

35

32

32

77

33

43

43

36

47

62

56

33

33

55

55

end

L

Y

Y

L

Y

Y

Y

L

L

Y

removed AA

tIPFLPVLPQkPGGVR

sLNAQPLGTDNTIENNTPTETNR

ySLNAQPLGTDNTIENNTPTETNR

sDLVTkEPLITPkR

ySDLVTkEPLITPkR

lSTQIkEGR

aDFPEANAIkkkFLFR

lADFPEANAIkkkFLFR

sTHTVcAIDR

sSkkPTFHNTAPSkTNVNVPR

sTLDSHSLkLQSGSkFVkIkPVNNLR

ySTLDSHSLkLQSGSkFVkIkPVNNLR

aSINTGTTVASkkASHkFR

yADTSTAANTNSTILR

yTSSSSSSSSPSSkESAPVFTSkELEVAR

hNRILR

sTkVPDNAPR

ySTkVPDNAPR

tTkSAkEGEENVER

yTTkSAkEGEENVER

identified sequence

a

Up (↑↑) and down (↓↓) regulation of the respective peptides in the icp55Δ mitochondria are indicated. (Only peptides with at least five-fold changes are considered) as well as amino acid start and end positions. For Icp55 processed peptide sequences, the removed amino acid is given. Lowercase amino-terminal amino acids and Lys residues indicate dimethyl labeling.

DLD2

MRPL11

3

STF1

2

4

ATP3

gene

1

no

Table 1. Summary of the Identified Already Known (left, 1−22) and Novel (right, 23−36) Icp55 Substratesa

Journal of Proteome Research Communication



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CONCLUSIONS In summary, our novel ChaFRADIC approach is a powerful tool for N-terminal proteomics. Our strategy is clearly less timeconsuming than established methods, and a smaller sample amount is required to identify (and quantify) a higher number of N-terminal peptides − albeit without the usage of glutamine cyclotransferase (Qcyclase) and pyroglutamyl aminopeptidase (pGAPase) to remove pyroglutamate, which is known to further improve the identification rate.17 Our method is easy to implement and robust; as we consider charge states +1 to >+4, it enables the identification of N-terminal peptides containing missed cleavage sites and His residues, which are usually lost in current COFRADIC approaches utilizing an initial SCX preenrichment of singly charged peptides to improve specificity.17 In contrast, specificities detected with our method were ∼80%. Moreover, ChaFRADIC can be readily combined with further proteases such as chymotrypsin or V8 because the labeled samples are pooled prior to digestion. We conclude that ChaFRADIC can be utilized for differential N-proteomics of limited sample amounts with unprecedented sensitivity (∼15 nonredundant N-terminal peptides quantified per microgram of sample). In principle, this will enable the usage of iTRAQ protein labeling27 instead of dimethyl labeling to compare differential proteolytic activity in up to eight different samples, as only a few micrograms per condition have to be labeled.

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diagonal chromatography; TAILS, terminal amine isotopic labeling of substrates

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(1) Blobel, C. P. Metalloprotease-disintegrins: links to cell adhesion and cleavage of TNF alpha and Notch. Cell 1997, 90 (4), 589−592. (2) Mossmann, D.; Meisinger, C.; Vogtle, F. N. Processing of mitochondrial presequences. Biochim. Biophys. Acta 2012, 1819 (9− 10), 1098−1106. (3) Vogtle, F. N.; Prinz, C.; Kellermann, J.; Lottspeich, F.; Pfanner, N.; Meisinger, C. Mitochondrial protein turnover: role of the precursor intermediate peptidase Oct1 in protein stabilization. Mol. Biol. Cell 2011, 22 (13), 2135−2143. (4) Blobel, G.; Dobberstein, B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 1975, 67 (3), 835−851. (5) Agard, N. J.; Wells, J. A. Methods for the proteomic identification of protease substrates. Curr. Opin. Chem. Biol. 2009, 13 (5−6), 503− 509. (6) Edman, P. A method for the determination of amino acid sequence in peptides. Arch. Biochem. 1949, 22 (3), 475. (7) Smith, H.; Edman, P.; Owen, J. A. N-Terminal amino-acids of human haptoglobins. Nature 1962, 193, 286−287. (8) Gevaert, K.; Goethals, M.; Martens, L.; Van Damme, J.; Staes, A.; Thomas, G. R.; Vandekerckhove, J. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted Nterminal peptides. Nat. Biotechnol. 2003, 21 (5), 566−569. (9) Kleifeld, O.; Doucet, A.; auf dem Keller, U.; Prudova, A.; Schilling, O.; Kainthan, R. K.; Starr, A. E.; Foster, L. J.; Kizhakkedathu, J. N.; Overall, C. M. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 2010, 28 (3), 281−288. (10) Foyn, H.; Van Damme, P.; Stove, S. I.; Glomnes, N.; Evjenth, R.; Gevaert, K.; Arnesen, T. Protein N-terminal acetyltransferases act as N-terminal propionyltransferases in vitro and in vivo. Mol. Cell. Proteomics 2013, 12 (1), 42−54. (11) Helsens, K.; Van Damme, P.; Degroeve, S.; Martens, L.; Arnesen, T.; Vandekerckhove, J.; Gevaert, K. Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation. J. Proteome Res. 2011, 10 (8), 3578−3589. (12) Prudova, A.; auf dem Keller, U.; Butler, G. S.; Overall, C. M. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics. Mol. Cell. Proteomics 2010, 9 (5), 894−911. (13) Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Phosphoproteomic analysis of the developing mouse brain. Mol. Cell. Proteomics 2004, 3 (11), 1093−1101. (14) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Stableisotope dimethyl labeling for quantitative proteomics. Anal. Chem. 2003, 75 (24), 6843−6852. (15) Mommen, G. P.; van de Waterbeemd, B.; Meiring, H. D.; Kersten, G.; Heck, A. J.; de Jong, A. P. Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO2-based depletion. Mol. Cell. Proteomics 2012, 11 (9), 832−842. (16) Vogtle, F. N.; Wortelkamp, S.; Zahedi, R. P.; Becker, D.; Leidhold, C.; Gevaert, K.; Kellermann, J.; Voos, W.; Sickmann, A.; Pfanner, N.; Meisinger, C. Global analysis of the mitochondrial Nproteome identifies a processing peptidase critical for protein stability. Cell 2009, 139 (2), 428−439. (17) Staes, A.; Impens, F.; Van Damme, P.; Ruttens, B.; Goethals, M.; Demol, H.; Timmerman, E.; Vandekerckhove, J.; Gevaert, K. Selecting protein N-terminal peptides by combined fractional diagonal chromatography. Nat. Protoc. 2011, 6 (8), 1130−1141. (18) Meisinger, C.; Sommer, T.; Pfanner, N. Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 2000, 287 (2), 339−342.

ASSOCIATED CONTENT

S Supporting Information *

List of the final protein N-termini and list of the nonredundant N-terminal peptides, which were identified and quantified, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, by the Bundesministerium fü r Bildung und Forschung (DYNAMO) and by the Deutsche Forschungsgemeinschaft (DFG ZA 639/1-1) is gratefully acknowledged. We furthermore would like to thank Julia Burkhart, Terkel Hansen, and Laxmikanth Kollipara for valuable discussions as well as Florian Beck and the PRIDE team. All data were uploaded to ProteomeXChange (http://www. proteomexchange.org, accession PXD000292) and can be inspected using PRIDE Inspector.28

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ABBREVIATIONS ACN, acetonitrile; ChaFRADIC, charge-based fractional diagonal chromatography; COFRADIC, combined fractional 3829



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(19) Jentoft, N.; Dearborn, D. G. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 1979, 254 (11), 4359−4365. (20) Burkhart, J. M.; Schumbrutzki, C.; Wortelkamp, S.; Sickmann, A.; Zahedi, R. P. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MSbased proteomics. J. Proteomics 2012, 75 (4), 1454−1462. (21) Burkhart, J. M.; Vaudel, M.; Gambaryan, S.; Radau, S.; Walter, U.; Martens, L.; Geiger, J.; Sickmann, A.; Zahedi, R. P. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012, 120 (15), e73−e82. (22) Staes, A.; Impens, F.; Van Damme, P.; Ruttens, B.; Goethals, M.; Demol, H.; Timmerman, E.; Vandekerckhove, J.; Gevaert, K. Selecting protein N-terminal peptides by combined fractional diagonal chromatography. Nat. Protoc. 2011, 6 (8), 1130−1141. (23) Thingholm, T. E.; Palmisano, G.; Kjeldsen, F.; Larsen, M. R. Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency. J. Proteome. Res. 2010, 9 (8), 4045−4052. (24) Spivak, M.; Weston, J.; Bottou, L.; Kall, L.; Noble, W. S. Improvements to the percolator algorithm for Peptide identification from shotgun proteomics data sets. J. Proteome Res. 2009, 8 (7), 3737− 3745. (25) 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 (3), M111 013722. (26) Craig, R.; Cortens, J. P.; Beavis, R. C. Open source system for analyzing, validating, and storing protein identification data. J. Proteome Res. 2004, 3 (6), 1234−1242. (27) Wiese, S.; Reidegeld, K. A.; Meyer, H. E.; Warscheid, B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 2007, 7 (3), 340−350. (28) Wang, R.; Fabregat, A.; Rios, D.; Ovelleiro, D.; Foster, J. M.; Cote, R. G.; Griss, J.; Csordas, A.; Perez-Riverol, Y.; Reisinger, F.; Hermjakob, H.; Martens, L.; Vizcaino, J. A. PRIDE Inspector: a tool to visualize and validate MS proteomics data. Nat. Biotechnol. 2012, 30 (2), 135−137.

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