Ultra Fast Trypsin Digestion of Proteins by High Intensity Focused

Proteolytic digestion of proteins in seconds under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) has been achieved. Success...
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Ultra Fast Trypsin Digestion of Proteins by High Intensity Focused Ultrasound D. Lo´ pez-Ferrer,† J. L. Capelo,*,‡ and J. Va´ zquez*,† Centro de Biologı´a Molecular “Severo Ochoa”-CSIC, 28049 Cantoblanco, Madrid, Spain, and REQUIMTE, Departamento de Quı´mica, Faculdade de Ciencia e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, 2829-516, Caparica, Portugal Received April 21, 2005

Proteolytic digestion of proteins in seconds under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) has been achieved. Successful in-solution and in-gel tryptic digestion of proteins in 60 s or less was demonstrated by either MALDI-TOF mass spectrometry or liquid chromatographyelectrospray ion trap mass spectrometry (RP-HPLC-ESI-IT-MS/MS). The efficiency of this new procedure for protein digestion compared favorably with those attained using conventional overnight incubation methods. The performance of the method was also demonstrated by the specific identification of three proteins in a whole proteome in less than 1 h. The method greately reduces the time needed for protein digestion, is of easy implementation, environmental friendly, and economic. Adaptation of this method to on-line procedures and robotic platforms could have promising applications in the proteomics field. Keywords: protein digestion • high-intensity focused ultrasound • MALDI • LC-ESI-IT-MS

Introduction The challenge of proteomics is to elucidate the physical organization, and to identify and quantitatively monitor the dynamics of proteomes in living organisms as efficiently as possible. Hence, high-throughput identification of proteins is mandatory; this task is generally achieved by combining protein or peptide separation techniques with identification/quantification with mass spectrometry. Complex mixtures of proteins are usually separated using one-dimensional or two-dimensional polyacrylamide gel electrophoresis (PAGE); alternatively, unseparated protein mixtures are digested and the peptide pool separated and analyzed on-line by two-dimensional liquid chromatography (2D-LC).1,2,3 In the case of PAGE, proteins are separated in bands or spots, which are excised and subjected to in-gel proteolytic digestion while in the case of 2D-LC, the enzymatic digestion is performed directly in solution. Digestion is usually performed with the proteolytic enzyme trypsin, although other enzymes such as Lys-C, Asp-N, or Glu-C have also been used for some purposes.4 Peptides are separated by chromatography, generally using a combination of strong cation exchange (SCX) separation followed by reversed-phase liquid chromatography,5,6 on-line with electrospray ionization and MS/MS analysis; the fragment mass spectra are then * To whom correspondence should be addressed: J. Va´zquez, Centro de Biologı´a Molecular Severo Ochoa, Facultad de Biolo´gicas, Universidad Auto´noma de Madrid, 28049 Cantoblanco, Madrid, Spain. Fax: 34 91 497 8087. E-mail: [email protected]. J. L. Capelo, Departamento de Quı´micaREQUIMTE, Faculdade de Cieˆncia e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre 2829-516, Caparica, Portugal. Phone + 351 212 949 649. Fax + 351 21 294 8550. E-mail: [email protected]. These two authors contributed equally to this work. † Centro de Biologı´a Molecular “Severo Ochoa”-CSIC. ‡ REQUIMTE, Departamento de Quı´mica, Faculdade de Ciencia e Tecnologia, Universidade Nova de Lisboa. 10.1021/pr050112v CCC: $30.25

 2005 American Chemical Society

correlated against protein databases using search engines such as Sequest7 or Mascot.8 Some routine methods have been proposed to evaluate results obtained from large-scale peptide identification studies.9 To facilitate protein degradation, reductive alkylation of proteins before enzymatic digestion is usually performed to achieve protein denaturation, removal of disulfide bonds, and alkylation of free -SH groups to prevent their reassociation. Typical steps involved in reductive alkylation include incubation of protein with a 40-fold molar excess of dithiothreitol in 8 M urea for 60 min at 37 °C, followed by the addition of excess alkylating agent, and a further incubation for 45 min.9 In-gel digestion handling is even more time-consuming,4 usually being performed overnight. Hence, the procedure for sample preparation plus trypsin digestion is slow and considerably limits the speed of protein identification. To date, different attempts have been made to reduce the time of sample preparation for protein identification; they are based on increasing temperature during digestion step,3 using columns containing immobilized trypsin,10 combinations of these,11 addition of organic solvents,1 or enhancing trypsin digestion by microwave energy.12 Properties of physical and chemical reactions are dramatically modified under the effect of an ultrasonic field generated by an ultrasonic probe (HighIntensity Focused Ultrasound, HIFU).13 Notably, enzymatic digestion in yeast for Se speciation has been enhanced in an ultrasonic field (ultrasonic probe) reducing the sample time from overnight (12 h) to seconds.14,15 Although the mechanism that is responsible for the enzymatic digestion enhancement using focused ultrasound is not completely understood, it appears to be related to an increase in diffusion rates as consequence of the cavitation phenomena and heating. Recent Journal of Proteome Research 2005, 4, 1569-1574

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research articles research in ultrasonic applications in medicine and drug delivery has estimated the pressure at the tip of the jet generated by bubble collapse (cavitation phenomena) around 60 MPa.16 This is high enough to penetrate small pores. Hence, liquid jets may act as microsyringes, delivering the enzyme to a region of interest, and they could be used for in-gel digestion of proteins. Furthermore, when a cavitating bubble collapses near the surface of a solid sample particle, micro-jets of solvent propagate toward the surface at velocities greater than 100 m s-1, and cause pitting and mechanical erosion of the solid surface, this leading to particle rupture (i.e., disruption);17 this process could be favorably used to enhance peptide release from the gel. In the present work, a very fast (60 s or less) and efficient in-solution or in-gel trypsin digestion of isolated proteins or whole proteomes is described. Digestion is performed under an ultrasonic field created by HIFU. The optimization of parameters affecting the process, such as extraction media, sample/enzyme ratio, sonication time, predigest ion preparation of sample and probe diameter is described. Results were compared with those obtained by classical overnight enzymatic digestion.

Materials and Methods Materials and Reagents. Protein molecular weighs standards were purchased from Amersham (Uppsala, Sweden). Sequence grade trypsin was obtained from Promega (Madison,WI). Dithiothretiol (DTT), iodoacetamide (IAA), ammonium bicarbonate (Ambic), acetic acid, and potassium phosphate salts were ordered from Sigma-Aldrich (St. Louis, MO). HPLC grade solvents were from LabScan (Dublin, Ireland). All gel electrophoresis reagents were obtained from Bio-Rad Laboratories, Inc (Hercules, CA). In-Solution Digestion. The conventional overnight digestion was performed during at least 12 h after denaturation with 8 M urea and reduction with 10 mM DTT in 25 mM ammonium bicarbonate pH 8.25 at 37 °C for 1 h. Iodoacetamide was then added to a final concentration of 50 mM. The resulting mixture was incubated at room temperature in darkness for 45 min. The mixture was then diluted 4-fold to reduce urea concentration, and after addition of of trypsin (1:50 protease-to-protein ratio), was incubated at 37 °C overnight. To set the technique up, 10 µgr of BSA was used as standard protein, and digestion was performed in a volume of 100 µL. In the case of proteome digestion, 100 µg of total cell extract were submitted to digestion procedures avoiding reduction and alkylation steps. High-Intensity Focused Ultrasound (HIFU)assisted digestion was performed in the same conditions as above, the final digestion volume was set to 100 µL and trypsin digestion was performed in 500 µL Eppendorf vials for 60 s (unless otherwise indicated) under sonication. A dr Hielscher (Teltow, germany) ultrasonic probe model 2000 (200 W) was used with the 1 or the 2 mm probe tips. The ultrasonic amplitude was set at 50% for all experiments. In-Gel Digestion. For in-gel digestion studies, 0.5 µg of different protein standards were loaded onto 10% SDS-PAGE gels. Coomasie Blue-stained protein bands were excised from the gels, cut into pieces and subjected to digestion according to the following procedures. In the conventional overnight digestion protocol, excised gel bands were washed with water, dehydrated with acetonitrile and dried in a vacuum centrifuge. Gel pieces were further rehydrated for 40 min in ice bath in a 0.5 µM solution of unmodified bovine trypsin in 25 mM 1570

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ammonium bicarbonate buffer, to a final volume of 12 µL. After the rehydration step, samples were digested overnight at 37 °C. Heat-enhanced digestion was performed using the same conditions, except that trypsin concentration was set to 1 µM, and digestion was carried out at 60 °C for 30 min, as described.3 HIFU-assisted trypstin digestion was performed during 60 s (unless otherwise indicated) following the conventional overnight digestion protocol, except that after rehydration, 100 µL of milli-Q water (with or without buffer) was added. Samples were sonicated at 50% amplitude with the 1 and 2 mm probe tips. Cell Culture and Protein Extraction of Endothelial Cells. Cells were grown at 37 °C, 5% CO2. The EA.hy926 cell line (kindly provided by Dr. Antonio Martinez-Ruiz) was cultured in DMEM with HAT supplement, 20% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 5 µg/mL gentamicin. Unless otherwise stated, for cell treatments, cells were washed with PBS. For preparing cell protein extracts, confluent cells were scraped and re-suspended in nondenaturing lysis solution (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.1 mM neocuproine, and 1% Triton X-100 plus protease inhibitor cocktail), incubated in ice for 15 min, and centrifuged at 10 000 × g, for 15 min at 4 °C. Supernatants were collected and protein quantified with the Bradford reagent (Bio-Rad). Cell Culture and Protein Extraction of Murine Splenic Lymphocytes. Briefly, Murine splenic lymphocytes preparations (kindly provided by Prof. Manuel Freire) were obtained by pressing a suspension of freshly excised and minced spleens in RPMI 1640 medium through a 65 µm-mesh stainless steel screen. The resulting suspension was treated with erythrocyte lysis buffer (0.75% NH4Cl, 0.21% Tris-HCl, pH 7.2), washed twice in RPMI 1640 medium, and grown for 16 h at 37 °C under a humidified 5% CO2 atm in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mL of penicillin and 100 µg/mL of streptomycin, in the presence of Con A (2.5 µg/mL) and IL-2 (10 U/mL). Isolation and purification of target proteins from murine splenic lymphocytes was as described elsewhere.18 The mixture was then analyzed by SDS-PAGE. Analysis by Mass Spectrometry. In the case of in-solution digestions, 0.5 µL of digestion solution (50 ng or 760 fmol of BSA) was analyzed. In the case of in-gel digestions, whole supernantants were dried down, and then resuspended in 5 µL of 30% ACN, 0.1% TFA; 0.5 µL were then analyzed by MALDI-TOF mass spectrometry using an Autoflex model of Bruker Daltonic (Bremen, Germany) equipped with a reflector and employing either 2,5-dihydroxybenzoic acid or R-cyano4-hydroxycinnamic acid as matrix and a Anchor-Chip surface target (Bruker Daltonic, Bremen, Germany). Peak identification and monoisotopic peptide mass assignation were automatically performed using Flexanalysis software (Bruker Daltonic, Bremen, Germany), using default parameters, and automatically used for database searches, without user intervention. Database searches were performed using MASCOT (Matrix Science, London, UK), against a NCBI nonredundant protein sequence database (www.ncbi.nih.gov),8 allowing two missed cleavages, and setting peptide tolerance to 75 ppm after close-external calibration. A match was considered successful when protein identification score is located out of the random region and the protein analyzed scores in first position. Five micrograms of peptide mixture were analyzed by RPHPLC on line with MS/MS detection as described.9 A Surveyor LC system was coupled to either a LCQ-DECA XP PLUS ion trap (Thermo-Finnigan, San Jose, CA), or to a LTQ linear ion

Ultra Fast Trypsin Digestion of Proteins by HIFU

Figure 1. MALDI-TOF mass spectra obtained after in-solution HIFU-assisted enzymatic cleavage of BSA in different conditions. a, ammonium bicarbonate buffer, pH 8.2; b, water; c, phosphate buffer, pH 7.8; d, water. In a, b, and c, BSA was reduced and alkylated prior to digestion, whereas in d no pretreatment was performed. Sonication time, amplitude and volume were 40 s, 50% and 115 µL, respectively. 1 mm probe was used. The number of tryptic peptides from BSA automatically detected were 8, 13, 9, and 11, respectively. For a better comparison of the results, the intensity scale in all the plots was set to 1/3 of the intensity of the highest peak.

trap (Thermo-Finnigan, San Jose, CA). The separation column was a 0.18 mm × 150 mm Biobasic RP column (ThermoHypersil-Keystone), and it was operated at 1.5 µL/min. Peptides were eluted using a 20 min gradient from 5% to 40% solvent B (Solvent A: 0.5% acetic acid. Solvent B: 0.5% acetic acid, 80% acetonitrile). MS/MS data were analyzed using Sequest, exactly as described.9

Results and Discussion Effect of HIFU Treatment on Enzymatic Protein Cleavage in Solution. Initial studies of HIFU-assisted enzymatic digestion of proteins were carried out using BSA as a model protein. These experiments were performed in different liquid media: (i) water, (ii) ammonium bicarbonate, and (iii) potassium phosphate buffers. Although it is well-known that enzymatic cleavage needs controlled pH conditions and hence should be done in a buffered media, previous research with HIFU and enzymes showed an unexpected enzymatic digestion of yeast material (for the quantification of Se species in biological samples, such as Se-methionine or Se-cysteine) with trypsin in a nonbuffered media.15 On the other hand, it has been described that bicarbonate and carbonate could act as hydroxyl radical scavengers.19 Radicals are formed when HIFU is applied to aqueous solutions and play an essential role in the enhancement of reaction rates.13 Hence, a phosphate buffer was also assessed. Figure 1 shows the MALDI-TOF mass spectra of the digested samples in the aforementioned conditions. As shown, a successful enzymatic digestion was attained in 40 s of HIFU treatment in all the cases. Digestion was slightly worse when performed in bicarbonate buffer, in comparison with that achieved by using phosphate, as judged by the corresponding peak intensities. Trypsin cleavage was also achieved in nonbuffered media without previous reduction and alkylation of the protein (Figure 1d). In all of the experimental conditions used in Figure 1, a correct protein identification in databases was achieved by peptide mass fingerprinting (not shown). In addition, these results, obtained by applying HIFU in only 40 s, were similar to those obtained using the classic method of

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Figure 2. MALDI-TOF mass spectra obtained after in-gel digestion of protein bands corresponding to BSA in different conditions. a, 12.5 mM ammonium bicarbonate media, 60 °C during 30 min, according to Havlis et al.3 b-d: HIFU-assisted digestion. The digestion was performed in either 12.5 mM ammonium bicarbonate (b), water (c) or water methanol, 80-20% v/v (d). HIFU conditions were: 115 µL sample volume, 60 s sonication time 50% amplitude and 2 mm sonication probe. Trypsin concentration was set to 0.75 µM in all cases.

overnight enzymatic digestion (data not shown; see below). When trypsin digestion was performed in the same conditions as above but without the HIFU treatment, no evidence of enzymatic digestion was observed in any case (not shown), indicating that reaction enhancement was directly related to HIFU treatment. Finally, when HIFU was applied to solutions containing BSA in the absence of trypsin, no evidence of protein degradation products was observed, indicating that HIFU treatment did not alter protein integrity (data not shown). Effect of HIFU Treatment on Enzymatic Protein Cleavage “in gel”. We next studied whether HIFU could also be applied to enzymatic cleavage of SDS-PAGE-separated protein bands. For this purpose, HIFU-assisted trypsin digestion of a set of identical bands from a Coomassie-stained SDS-PAGE gel, corresponding to BSA each, was performed during 60 s in (i) ammonium bicarbonate buffer, (ii) water, and (iii) a 80:20 mixture of water-methanol (v/v). The latter medium was chosen because Rusell et al.1 reported an increase in peptide release from gel slides when the extraction was performed in this solvent. In addition, to compare the HIFU digestion with a more conventional approach, slides of the same sample were also subjected to the Havlis et al.3 digestion protocol, performed during 30 min at 60 °C. Results are shown in Figure 2. For Havlis’ and HIFU-assisted methods (Figure 2a,b), comparable signal-to-noise ratios and intensities were observed; nine peptides were automatically identified in both cases. Both methods allowed a positive protein identification in databases by peptide mass fingerprinting with a MASCOT score of 69 and 87, respectively. The digestion performance was clearly lower when the treatment was applied in water alone or water/ methanol (Figure 2c,d), and protein identification was not possible in these conditions. These results indicated that by applying a HIFU treatment in appropriate conditions, digestion yields comparable to those obtained by previously published methods could be attained in only 60 s. Additional studies were conducted to compare the extent of tryptic peptides generation by two different ultrasonic probes. The probe’s size is limited by the sample volume to be treated with HIFU. Since the total volume in which HIFU Journal of Proteome Research • Vol. 4, No. 5, 2005 1571

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Lo´ pez-Ferrer et al. Table 1. Sequence Coverage (%), Matched Masses and Mascot Score of Digestion Products of a Lysozyme Gel Slide after In-Gel HIFU Digestion at Different Treatment Timesa HIFU time (seg)

sequence coverage (%)

matched masses

Mascot score

10 40 60 80 120

57 59 44 56 47

8 10 9 8 12

104 130 98 100 116

a Incubation times used were (a) 10, (b) 40, (c) 60, (d) 80, and (e) 120 s. Enzyme concentration was 0.75 µM in all cases.

Figure 3. “Dead zones” in ultrasonic treatments. An 500-µL eppendorf tube was shown containing a piece of SDS-PAGE gel and 115 µL digestion solution.

gel digestions were developed was 115 µL, the diameter of the probes was limited to 1 and 2 mm. During the course of these experiments, we observed that it was of paramount importance to maintain the gel slide under the ultrasonic field generated by the probe. The existence of the so-called “dead zone”, i.e., the zone where cavitation is not achieved, is one of the most important factors to consider when using probe sonication. The distribution of ultrasonic waves is an important parameter to be taken into account for the process optimization, since variations of the local cavitational activity and the resulting pressure field as a function of axial and radial distance from the horn tip has been demonstrated.20,21 The high energy density produced from a single probe tip is unlikely to be capable of delivering enough energy density to affect the whole of a large reacting volume. Hence, the distance between the horn tip and the wall container must be as short as possible. Eppendorf-type vessels should be employed, since the small diameter raises the liquid level of the sample without increasing the volume, thereby allowing the probe to be inserted deep enough into the solution. As can be seen in Figure 3, when the 1 mm probe was used, the gel pieces tended to reach the “dead zones” of the ultrasonic probe, where the cavitation effects are negligible,13 and the pieces remained in such place during all the process. Under these conditions, no appreciable digestion was detected (data not shown). This problem was not observed with the 2 mm probe, since the excised gel had not enough free space to reach the dead zone, remaining fully exposed to the ultrasonic field during treatment (Figure 3b); under these conditions, trypsin digestion was successfully attained. In another experiment, the same treatment was applied to the sample but a water bath at 60 °C and 4 min shaking was 1572

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Figure 4. Analysis of HIFU-assisted digestion performance as a function of trypsin concentration. HFU-assisted in gel trypsin digestion of a gel slide containing ovalbumin was performed in standard conditions, but setting trypsin concentration at: (a) 0.5 µM (b) 1.5 µM, and (c) 3 µM. Peaks identified as proteolytic products from ovalbumin are indicated by an asterisk; autolysis peptides from trypsin are indicated by a T.

used instead of ultrasonication. No peptide fragments could be detected by MALDI-TOF MS in these conditions (not shown). Finally, no protein degradation was observed when ultrasonication was applied to the protein band in absence of trypsin. Hence, it was confirmed that HIFU treatment only assists and enhances the enzymatic digestion of proteins and peptide release from gel, without affecting protein integrity. Effect of HIFU Treatment Time, Trypsin Concentration and Organic Solvent. Results summarized in Table 1 show the MALDI-TOF MS data obtained for the HIFU-assisted digestion of a lysozyme gel bands using different incubation times. Interestingly, the signal-to noise ratio was virtually the same regardless the sonication time used (range 10-120 s) (not shown). Surprisingly, the lowest time used, 10 s, was enough to promote tryptic digestion and protein identification. As depicted in Table 1, no significant differences in protein identification parameters were evident. Therefore, HIFU greatly accelerates the digestion process, providing good sequence coverage of the protein even in seconds; in clear contrast, the classic method provides no detectable cleavage products in the

Ultra Fast Trypsin Digestion of Proteins by HIFU

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Figure 5. MALDI-TOF mass spectrometry comparison of HIFU-assisted digestion performance with that achieved by conventional approaches. Three identical protein bands containing ovalbuming were subjected to (a) conventional overnight digestion (12 h), (b) 30 min digestion, according to Havlis et al.,3 and (c) HIFU-assisted digestion (60 s).

same time range (not shown). A time of 60 s was chosen as the standard procedure for further experiences. In a parallel set of experiments, the influence of trypsin concentration on the performance of HIFU-assisted enzymatic in gel protein digestion was analyzed. As shown in Figure 4, where ovalbumin gel bands were subjected to digestion, variation of enzyme concentration did not have an appreciable influence on digestion yields. To analyze the effect of the presence of organic solvent, gel spots containing identical protein bands were also subjected to the following digestion protocols: (i) 60 °C, 30 min digestion,3 (ii) HIFU-assisted digestion, and (iii) HIFU-assisted digestion in the presence of a 80:20 (v/v) mixture of water and acetonitrile.1 Protein identification was successfully achieved following the 60 °C, 30 min digestion, and HIFU protocols, attaining a protein sequence coverage of 55.4% and 51.7%, respectively. The presence of acetonitrile did not improve the recovery yield and the protein coverage attained was lower, 37.7%, although protein identification in databases was also possible. As a general trend, organics seems to act as scavengers of ultrasonic efficiency. Hence, organics should be avoided when HIFU is applied to protein digestion. Comparison with Conventional Digestion Procedures. The digestion performance of the HIFU-assisted, 60 s digestion protocol of a SDS-PAGE gel band containing ovalbumin was compared with that achieved by conventional overnight incubation. The 30 min, 60 °C protocol of Havlis et al.3 was also compared in this experiment. Results are presented in Figure 5. The number of automatically idenfied peptides, Mascot scores and sequence coverages were similar in the three cases (9/100/48%, 10/113/43%, and 9/97/42%, for conditions a, b, and c in Figure 5, respectively). These results demonstrate that using the HIFU-assisted digestion methodology the same performance was achieved than with the classical approaches, but with a considerable reduction in the sample handling and manipulation time. The comparative performance of the conventional, overnight method was also compared with that of the 60 s, HIFU-assisted method by applying both methods to the analysis of identical protein bands from a complex biological sample, separated by SDS-PAGE. For this purpose, a 60 kDa slide was analyzed by both protocols. As shown in Figure 6, the two protocols yielded MALDI-TOF mass spectra with a similar quality and protein sequence coverage. Application to the Analysis of Complex Peptide Mixtures by LC-ESI-MS/MS. To analyze the usefulness of this method for high-speed, specific protein identification by HPLC online with ESI and MS/MS analysis, a total proteome extract from a preparation of endothelial cells was separated in two identical aliquots and subjected to trypsin digestion either following the conventional overnight approach or by HIFU-assisted digestion

Figure 6. MALDI-TOF mass spectrometry comparison of HIFUassisted digestion performance with that achieved by conventional overnight incubation. A preparation of murine splenic lymphocytes was subjected to SDS-PAGE analysis, and a band at around 60 kDa was scised from the gel, cut in two pieces and subjected to either (a) HIFU-assisted digestion (60 s) or (b) conventional overnight digestion (12 h). The band corresponded to human glutamate dehydrogenase-Apo form; peaks identified as proteolytic products from this protein are indicated by an asterisk. These peaks gave a sequence coverage of 51% in a) and 44% in (b).

for only 60 s. The digested peptide mixtures were analyzed by RP-HPLC on line with linear ion trap mass spectrometry analysis, using a 40 min gradient, performing a full scan followed by three MS/MS scans on the most intense ions using dynamic exclusion and six MS/MS scans pre-programmed on three ions corresponding to peptides known to derive from the proteins Vimentin, Annexin A2, and Histone h2Am. As shown in Figure 7, upper panels, the full scan intensity base peak trace of peptides were similar in both cases, suggesting a similar digestion yield, although the 60 s, HIFU-assisted method produced slightly higher-intensity peaks. We observed that the total number of identified peptides was somewhat higher using the conventional protocol than that obtained by the HIFU approach; similarly, the number of cleavages missed by trypsin was also significantly higher for the HIFU-based protocol. However, the specific selected ion monitoring trace of the three peptides produced peaks whose intensity was higher when using the HIFU-based approach (Figure 7), although the Journal of Proteome Research • Vol. 4, No. 5, 2005 1573

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incubation, when the procedure was applied to single proteins or complex peptide mixtures, when the analysis was performed by either MALDI-TOF mass spectrometry or HPLC on-line with MS/MS analysis. High sample throughput can nowadays be achieved with the recently developed multi-probe sonicators.22,23 Hence, a minimum of 12 samples can be treated at once and protein digestion can be performed in seconds, thus allowing an easy implementation of this procedure for robotic platforms for enzymatic digestion. The possibilities of high sample throughput along with its easy implementation in robotic devices gives to the procedure here described an excellent future in biotechnology industry and proteome research.

Acknowledgment. This work was supported by grants CICYT BIO2003-01805, CAM 08.5/0065.1/2001, and CAM GR/ SAL/0141/2004 and by an institutional grant by Fundacio´n Ramo´n Areces to CBMSO. Authors want to acknowledge Dr. Anabel Marina and the members of the Proteomics Service of the Centro de Biologı´a Molecular Severo Ochoa for their helpful assistance and suggestions. The research findings here reported are protected by international laws under patent request PORT no. 23848 from the INI, Instituto Nacional da Propiedade Industrial from Portugal. Figure 7. Ultrafast specific identification of three model proteins (Vimentin, Annexin A2, Histone H2AM) in a whole proteome by HIFU-assisted trypsin digestion followed by RP-HPLC-MS/MS and monitorization of peptides specific for each protein. A crude protein extract from endothelial cells was divided in two identical aliquots, and subjected to trypsin digestion. Results obtained by using HIFU-assited, 60 s digestion method (left panels) were compared with those obtained by using the conventional overnight digestion procedure (right panels). Upper panel: base peak trace of ion species eluting from the HPLC column. Lower panels: intensity of peptides, SLYASSPGGVYATR, GVDEVTINLLTNR, and VTIAQGGVLPNIQAVLLPK from the above-mentioned proteins, obtained by SIR analysis. Insets show the corresponding MS/MS spectra (relative intensity vs m/z) at the peak apex; asterisks mark fragment peaks matching theoretical ions masses from y or b-series.

presence of these proteins in the whole proteome digest could be demonstrated in both cases. These findings suggest that although results were, in general, similar, trypsin digestion was not completely comparable in both protocols. Taken together, all of these results demonstrate that our method allows the specific identification of a protein in a whole proteome in less than 50 min (1 min incubation and 40 min HPLC gradient), with a performance similar to that obtained by the conventional overnight digestion procedure.

Conclusions Our results demonstrate that enzymatic in gel or in solution digestion of proteins in the second time range may be dramatically enhanced by performing the process under an ultrasonic field provided by High Intensity Focused Ultrasound technology. HIFU considerably simplifies and accelerates the sample preparation routine without compromising the yield of digestion products, sensitivity of peptide detection, and performance of protein identification. The new methodology provides similar results than conventional approaches based on overnight

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