A Clean, More Efficient Method for In-Solution Digestion of Protein

Abstract: Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of...
12 downloads 0 Views 258KB Size
Download PDF
A Clean, More Efficient Method for In-Solution Digestion of Protein Mixtures without Detergent or Urea Sung Chan Kim, Yue Chen, Shama Mirza, Yingda Xu, Jaeick Lee, Pingsheng Liu, and Yingming Zhao* Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038 Received July 11, 2006

Abstract: Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/ MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies. Keywords: in-solution digestion • proteomics • IMAC • phosphopeptides • adiposomes

Introduction Tandem mass spectrometry (MS/MS) combined with either reversed phase HPLC or multiple dimensional LC has become an indispensable tool for identifying and quantifying proteins in protein mixtures.1-3 These techniques have been used to profile protein complexes, organelles, and whole-cell lysates. To be successful, this approach to analyzing complex protein mixtures requires efficient digestion of the sample by a protease such as trypsin. In a typical experiment, the protein mixture of interest is denatured in 8 M urea, alkylated, and then diluted * Corresponding author. E-mail: [email protected], Fax: (214) 648-2797; Tel: (214) 648-7947.

3446

Journal of Proteome Research 2006, 5, 3446-3452

Published on Web 11/10/2006

to 1-2 M urea. The resulting sample is digested with trypsin. This strategy has been used to digest highly complicated protein mixtures, such as yeast whole cell lysate.2 However, salts and urea present in the resulting digest complicate subsequent analysis. Several different approaches for in-solution digestion avoiding use of chemical denaturants such as urea or SDS to increase digestion efficiency with simple protein mixtures or cell organelles have been reported. These methods include the use of thermal denaturation of proteins prior to in-solution digestion,4-5 trypsin digestion in organic solvents,6 and the use of microwave irradiation.7 Other in-solution digestion methods using CNBr/trypsin or surfactant such as SDS or n-octyl glucoside (n-OG) have been developed to efficiently digest highly hydrophobic samples such as membrane proteins or lipid rafts.8-11 These methods have successfully shown fast, efficient digestion of simple protein mixtures and are useful for MALDI-MS analysis. However, these methods might be not suitable for large-scale protein identification or global identification of post-translationally modified peptides, especially when highly complex protein mixtures such as whole cell lysates are applied. Because strong surfactants such as urea or SDS are commonly used to prepare whole cell lysates, these MS-unfriendly contaminants must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we report a cleaner procedure for in-solution digestion of complicated protein mixtures such as whole-cell lysates. This method involves precipitating the protein mixture of interest with trichloroacetic acid/acetone (TCA/acetone), which denatures the proteins and removes all the salts in the sample. The proteins are then partially resolubilized in NH4HCO3 buffer and digested with trypsin. After reduction of the disulfide bonds and alkylating cysteine residues, more trypsin is added to ensure complete digestion. We describe analysis of two complex mixtures using this procedure: whole cell lysate from A431 cells, and the phosphoproteome of HeLa cell adiposomes. We expect this digestion procedure will find applications in shotgun proteomics and large-scale analysis of protein phosphorylation by IMAC.

Experimental Section Materials. Fetal bovine serum (FBS), trypsin, and Dulbecco’s modified Eagle’s medium (DMEM) were from Life Technologies 10.1021/pr0603396 CCC: $33.50

 2006 American Chemical Society

technical notes Inc. (Carlsbad, CA). Dulbecco’s phosphate buffered saline (PBS) was from Sigma (St. Louis, MO). Urea, thiourea, CHAPS, ammonium bicarbonate, and dithiothreitol were from Fisher Scientific Corp. (Pittsburgh, PA). Colloidal Blue Staining Kit was from Invitrogen (Carlsad, CA). Sequencing-grade trypsin was from Promega (Madison, WI). µC18 ZipTips were from Millipore Corp. (Bedford, MA), and protease inhibitor mixture was from Roche (Indianapolis, IN). Preparation of Cell Lysate from A431 Cells. A431 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. After they reached confluence, the cells were washed twice with cold Dulbecco’s PBS, and then 400 µL of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM TrisHCl, pH 8.5, and protease inhibitor mixture) was added. The cell lysate was harvested and sonicated 3 times for 10 s each with 30-s intervals between sonications. The lysate was centrifuged at 22000g for 1 h. The pellet was discarded, and 5 mg of the cellular lysate was precipitated with TCA/acetone to denature whole proteins. Briefly, 1 volume of protein sample was mixed with a solution containing 1 volume of TCA and 8 volumes of acetone. The resulting solution was mixed and kept at -20 °C for 2 h. The protein pellet was recovered by centrifugation at 22000g for 10 min. The pellet was rinsed twice with cold acetone to remove residual salts. Isolation of HeLa Cell Adiposomal Proteins. HeLa cells were cultured in DMEM (high glucose) with 10% fetal bovine serum, 1% penicillin/streptomycin. HeLa cells were incubated with 100 µM oleate overnight prior to harvesting. Lipid droplets/adiposomes were purified by a modification of the method of Liu et al.12 Briefly, confluent cells from 40 × 150 mm plates were scraped into ice-cold PBS with proteinase inhibitor PMSF, resuspended in buffer A (25 mM tricine, pH 7.6, 250 mM sucrose, 0.2 mM PMSF), and homogenized by N2 cavitation (450 psi for 15 min on ice). The postnuclear supernatant (PNS) fraction (11 mL) was obtained by centrifugation at 1000g and loaded into a SW41 tube. The sample was centrifuged at 274000g for 1 h at 4 °C. The white band containing lipid droplets at top of gradient was collected in 0.5 mL and resuspended in 6 mL of buffer A in a SW41 tube. The droplet/ adiposome fraction was overlaid with 4 mL of buffer B (20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM MgCl2), and the sample was centrifuged at 274000g for 1 h at 4 °C. The droplet band at the top of gradient was collected in 0.5 mL. Then the sample was centrifuged at 20000g for 4 min and the procedure repeated four times. An additional wash in 1 mL of buffer B using a 265000g centrifugation in a TLA 100.3 tube for 5 min was applied to remove contaminating membranes. The adiposome proteins were precipitated by TCA/acetone. Digestion in the Presence of Urea or SDS. Five miligrams of the TCA/acetone-precipitated and denatured pellet was resolubilized in 1 mL of 100 mM NH4HCO3 (pH 8.0)/8 M urea or 100 mM NH4HCO3 (pH 8.0)/2% SDS. The resolubilized proteins were reduced with 5 mM dithiothreitol (50 °C, 30 min) and then alkylated with 15 mM iodoacetamide at room temperature for 30 min in the dark. The reaction was quenched with 15 mM cysteine at r.t. for 30 min. Before tryptic digestion, 100 mM ammonium bicarbonate buffer was added to reduce the concentration of urea or SDS to 1 M or 0.1%, respectively. For in-solution digestion, trypsin was added to the protein mixture at an enzyme-to-substrate ratio of 1:50 (w/w). After incubation at 37 °C for 16 h, additional trypsin (1:100, w/w) was added to the sample and incubation was continued for 3 h to ensure complete digestion. Forty micrograms of the

Kim et al.

resulting tryptic peptides or undigested proteins was subjected to SDS-PAGE followed by colloidal blue staining. µC18 ZipTips were used to wash the tryptic peptides according to the manufacturer’s directions before nano-HPLC/MS/MS analysis. Clean Digestion in NH4HCO3 Buffer. Five miligrams of the TCA/acetone-precipitated and denatured pellet was resuspended in 1 mL of 100 mM NH4HCO3 (pH 8.0), resulting in only partial solubilization of the peptides. The protein pellet resuspended by NH4HCO3 was ground by a round glass rod in a microcentrifuge tube and sonicated three times for 20 s each with 30-s intervals between sonications to make a homogeneous protein suspension. For in-solution digestion, trypsin was added to the protein mixture at an enzyme-to-substrate ratio of 1:50 (w/w). After incubating at 37 °C for 16 h, the tryptic peptides were reduced and alkylated as described above. Additional trypsin (1:100 w/w) was added, and the mixture was incubated at 37 °C for 3 h to ensure complete digestion. Forty micrograms of the resulting tryptic peptides or undigested proteins were subjected to SDS-PAGE followed by colloidal blue staining. µC18 ZipTips were used to clean the tryptic peptides before nano-HPLC/MS/MS analysis. Methylation of Adiposomal Proteins. Methylation of carboxylic groups of acidic amino acid residues (D and E) and the C-termini of peptides was carried out using a procedure previously described.13 Briefly, 100 µg of tryptic digest was dried in a SpeedVac (Thermo Savant, San Jose, CA) for 6 h. Then 50 µL of 2 M methanolic HCl was added to the dried peptides, and the reaction was carried out for 2 h on a shaker. The anhydrous methanol used for the preparation of methanolic HCl had been distilled against CaH2 by a standard method. The peptide mixture was thoroughly dried, and the methylation reaction was repeated to ensure complete conversion of carboxylic acid groups to their corresponding methyl esters. Batchwise IMAC. Batchwise IMAC was carried out according to an optimized procedure. First, Poros 20 MC beads were activated according to the manufacturer’s instructions. A 10µL bed volume of activated beads was equilibrated with 200 µL of loading solution [acetonitrile/methanol/water (1:1:1, v/v/ v) in 0.1% acetic acid] prior to sample loading. One hundred micrograms of methylated adiposomal tryptic peptides was dissolved in 20 µL of acetonitrile/methanol/water (1:1:1, v/v/ v), and the pH was adjusted to 2.5-3.0 with 2 M NH4HCO3 (pH 8.0). The peptide mixture was then centrifuged at 100000g for 20 min to remove insoluble particles. The resulting supernatant was mixed with IMAC beads and incubated at room temperature with shaking for 30 min. After incubation, the suspension was centrifuged in a microcentrifuge at 13000g for 1 min, and the supernatant was removed. The beads were washed once with 200 µL of washing buffer I [acetonitrile/ methanol/water/acetic acid (75:10:14:1, v/v/v/v) to which was added NaCl to 100 mM], followed by two washes with 200 µL of washing buffer II [acetonitrile/water/acetic acid (85:14:1, v/v/ v)]. Bound phosphopeptides were eluted three times from the IMAC beads with 50 µL of elution solution [acetonitrile/water/ trifluoroacetic acid (45/50/5, v/v/v)]. Nano-HPLC/MS/MS Analysis. HPLC/MS/MS analysis was performed in an LCQ DECA XP ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a nano-ESI source. The electrospray source was coupled online with an Agilent 1100 series nano flow LC system (Agilent, Palo Alto, CA). Two microliters of the peptide solution in loading buffer (2% acetonitrile/97.9% water/0.1% acetic acid (v/v/v)) was manually injected and separated in a nano-HPLC column (50 mm length Journal of Proteome Research • Vol. 5, No. 12, 2006 3447

Clean In-Solution Digestion of Protein Mixtures

× 75 µm ID, 5 µm particle size, 300 Å pore diameter) packed in-house with Luna C18 resin (Phenomenex, St. Torrance, CA). Peptides were eluted from the column with a gradient of 5% to 80% buffer B (90% acetonitrile/9.9% water/0.1% acetic acid (v/v/v)) in buffer A (2% acetonitrile/97.9% water/0.1% acetic acid (v/v/v)) over 10 min. Eluted peptides were electrosprayed directly into the mass spectrometer. MS/MS spectra were acquired in a data-dependent mode, in which the two strongest ions in each MS spectrum were selected for fragmentation. Mass Spectrometry and Protein Sequence Database Searching To Identify Phosphoproteins. Nano-HPLC/LTQ mass spectrometry was carried out as previously described14 except that a 9-min gradient of 6-90% B buffer (90% acetonitrile, 9.95% water, 0.05% acetic acid) in A buffer (97.95% water, 2% acetonitrile, 0.05% acetic acid) at a low flow rate of 0.1 µL/min was used. The LTQ was operated in a data-dependent mode where one full MS scan was followed by four pairs of MS2/ MS3 scans. MS3 was automatically triggered when a neutral loss peak of 98, 49 or 32.7 ((2) m/z was detected among the top eight most intense peaks in the MS2 spectrum. MS3 fragmentation on the dominant neutral loss ions generated a rich backbone fragmentation pattern for correct peptide identification and accurate phosphorylation site determination. Normalized collision energy was set to 22% during MS2 acquisition and 35% during MS3 acquisition. Protein Sequence Database Searching and Manual Verification. Tandem mass spectra were used to search the NCBInr database with the Mascot search engine (version 2.0, Matrix Science, London, UK). Mass tolerance was set to (4 Da for parent ion masses and (0.6 Da for fragment ion masses. Peptides with Mascot scores above 40 were considered potential positive identifications and were manually verified. Strict manual analysis was applied to validate protein identification results, using the following criteria. y, b, and a ions as well as their water loss or amine loss peaks were considered. For doubly charged or triply charged ions, all the major isotopically resolved peaks matched fragment masses of the identified peptide. The isotopically resolved peaks were emphasized because single peaks can come from electronic sparks and are less likely to be relevant to peptide fragments. The major isotopically resolved peaks for doubly charged or triply charged ions were defined as (1) those isotopically resolved daughter ions with m/z higher than parent m/z and intensity higher than 5% of the maximum intensity or (2) those isotopically resolved peaks with intensities higher than 20% of the maximum intensity and m/z values between one-third of the parent m/z and the parent m/z. Typically, more than seven independent (amine and water loss not counted), isotopically resolved peaks were matched to theoretical masses of the peptide fragments. For manual validation of singly charged parent ions, the Mascot score was required to be equal to or above the identity score threshold of the peptide. For peptides ending with arginine or lysine residues, at least four consecutive amino acids in the peptide sequence had to be confirmed with the isotopically resolved b-ion and the y-ion series.

Results A Clean Method To Digest Proteins in NH4HCO3 Buffer. Tryptic digests are usually performed in a reaction buffer such as NH4HCO3 to produce tryptic peptides without contamination from detergents, urea, or salts. For example, NH4HCO3 buffer is routinely used for in-gel digestion. However, NH4HCO3 buffer is intrinsically unable to extract proteins from either organelles 3448

Journal of Proteome Research • Vol. 5, No. 12, 2006

technical notes

Figure 1. Digestion of suspended proteins by trypsin. (A) Photographs of protein sample during the course of tryptic digestion at 0, 1, and 16 h. Five milligrams of the TCA/acetoneprecipitated and denatured protein from A431 whole-cell lysate was resuspended in 1 mL of 100 mM NH4HCO3. The resulting suspension was digested with trypsin at an enzyme-to-substrate ratio of 1:50 (w/w). (B) SDS-polyacrylamide gel, stained with colloidal blue, showing protein samples before and after 16-h digestion with trypsin (enzyme-to-substrate ratio 1:50, w/w). Forty micrograms of protein or tryptic peptides was loaded onto a 12% SDS-PAGE gel. U: trypsinization in the presence of 1 M urea; S: trypsinization in the presence of 0.1% SDS; N: trypsinization in the presence of 100 mM NH4HCO3.

or cells. Instead, proteins are typically extracted with buffer containing detergent or urea, which has the additional effect of denaturing the proteins. The resulting solution may then be diluted 4- to 8-fold with buffer to reduce the denaturant concentration to a level at which trypsin is active. A problem with this procedure is that the presence of urea or detergents is not desirable for HPLC/mass spectrometric analysis, or for methylation of carboxylic acids (as is commonly done when IMAC is used to isolate phosphopeptides). In principle, this problem can be solved by precipitating the protein extract with TCA/acetone to remove denaturants and resuspending in buffer amenable to subsequent analysis. Unfortunately, NH4HCO3 buffer, the buffer of choice for tryptic digests, does not completely solubilize TCA/acetone protein pellets. To our knowledge, NH4HCO3 buffer has not been used to resuspend protein pellets for in-solution digestion of protein mixtures before proteomics analysis. To test if protein pellets that have been partially solubilized in NH4HCO3 buffer can be digested efficiently, 100 mM NH4HCO3 (pH 8.0) was added to the denatured protein pellet obtained from TCA/acetone precipitation of the whole lysate from A431 cells. As expected, the denatured protein pellet could not be completely dissolved; small protein particles remained visible in a cloudy suspension (Figure 1A), suggesting incomplete dissolution. During the course of tryptic digestion, the suspended particles gradually disappeared (Figure 1A). Because tryptic peptides are likely to be more soluble than the original proteins, this observation suggests that proteolysis was pro-

technical notes

Kim et al.

Figure 2. Nano-HPLC/MS/MS analysis of tryptic peptides from digestion of A431 whole-cell lysate in the presence of urea or NH4HCO3. (A and D) Total ion chromatograms; (B and E) mass spectra obtained at retention times of 55.81 and 29.29 min, respectively; (C and F) tandem mass spectra of peaks at m/z 803.7 (at retention time 55.81 min) and 480.8 (at retention time 29.29 min), identified as peptides VVLAYEPVWAIGTGK from triosephosphate isomerase 1 and LNVTEQEK from enolase 1, respectively.

ceeding. After 16 h of digestion, the solution was transparent (Figure 1A). This result is consistent with previous report that aggregated protein pellets generated by thermal denaturation were easily digested and disappeared after trypsin digestion for 3 h and resulted in more specific cleavage and generation of sufficient numbers of tryptic peptides for protein identification.4 This result suggests that the denatured protein particles produced by TCA/acetone precipitation can be easily digested by trypsin in aqueous solution without any detergents as shown in Figure 1. In a parallel experiment, the same amount of protein pellet from A431 cells was solubilized in either urea or SDS buffer, diluted, and subsequently digested with trypsin. When samples from digestion reactions containing 1 M urea, 0.1% SDS, or 100 mM NH4HCO3 were analyzed by SDS-PAGE, proteins were nearly undetectable using Coomassie blue staining (Figure 1B), indicating that trypsin could digest proteins in all three solutions.

To check the degree of digestion, tryptic peptides from samples digested in the presence of urea or NH4HCO3 were analyzed exhaustively by nano-HPLC/LCQ mass spectrometry (Figure 2). Tryptic peptides from the digestion containing SDS were not analyzed by mass spectrometry due to the difficulty of efficiently removing SDS from the peptides. Mass spectrometric analysis of tryptic peptides from NH4HCO3-buffered digestion in combination with automated protein sequence database searching and manual verification led to the identification of 652 tryptic peptides, of which 631 (96.8%) were completely digested, 20 (3.1%) contained 1 missed cleavage site, and 1 (0.1%) contained 2 missed cleavage sites (Figure 3). Analysis of peptides digested in the presence of urea identified 664 peptides, of which 580 (87.3%) were completely digested, 81 (12.1%) contained 1 missed cleavage site, and 3 (0.5%) contained 2 missed cleavage sites (Figure 3). Our results suggest that proteins can be completely digested despite only partial solubilization in NH4HCO3 buffer. It was Journal of Proteome Research • Vol. 5, No. 12, 2006 3449

Clean In-Solution Digestion of Protein Mixtures

technical notes

Figure 4. Nature of amino acids surrounding the non-C-terminal arginine and lysine residues in peptides with missed cleavage sites shown in Figure 3.

Figure 3. Distribution of identified tryptic peptides from proteins resolubilized by urea or resuspended by NH4HCO3 buffer. (A) Total number of identified peptides and proteins from digestion in the presence of urea and NH4HCO3. Tryptic peptides from 2 µg of protein from A431 whole-cell lysate were analyzed by nanoHPLC/mass spectrometry. (B and C) Pie charts showing the distribution of tryptic peptides with 0, 1, and 2 missed cleavage sites.

surprising to observe that digestion of suspended proteins in NH4HCO3 buffer was more complete than digestion of fully dissolved proteins in dilute urea buffer. The difference in degree of digestion might arise from the renaturing of proteins in 1 M urea; the resulting folded proteins could be more resistant to enzymatic digestion. Alternatively, 1 M urea might lower the enzymatic activity of trypsin due to low-level inhibition15 or denaturation of the enzyme. Systematic Analysis of Incompletely Digested Peptides. We analyzed the amino acid residues surrounding the non-Cterminal arginine and lysine residues in tryptic peptides containing missed cleavage sites. A high proportion of arginine and lysine residues at which cleavage was missed were adjacent to an acidic residue (38% for urea digestion and 41% for NH4HCO3 digestion, Figure 4). Adjacent acidic residues might interact with the basic side chains, interfering with the binding of trypsin to lysine and arginine. Amino acid residues with potential to sterically hinder trypsin binding, such as valine 3450

Journal of Proteome Research • Vol. 5, No. 12, 2006

and isoleucine, were also commonly observed adjacent to missed cleavage sites. Application of the Clean Digestion Protocol to Subproteome Analysis. A clean digestion procedure can streamline sample preparation. Eliminating the need for desalting and buffer exchange increases throughput while reducing sample loss and contamination associated with sample handling. The net result should be increased sensitivity and improved sample integrity at the LC-MS/MS stage. To test our optimized digestion procedure, we applied it in conjunction with phosphopeptide enrichment through batchwise IMAC to the analysis of the adiposome phosphoproteome of HeLa cells. The adiposome is a metabolically active cellular lipid-storage organelle.12 This dynamic organelle has been implicated in such processes as lipid and sterol metabolism and membrane transport pathways. Consistent with these proposed functions, our proteomic screen of adiposomal phosphoproteins identified 7 categories of proteins, including those involved in fatty acid metabolism and signal transduction as well as protein and vesicle trafficking. In all, 33 phosphorylation sites were identified in 32 peptides representing 28 proteins (Table 1). Several observed phosphorylation sites were within known consensus sequence motifs. Two sites conformed to the calcium/calmodulin-dependent kinase (CAMK) motif. This result is reasonable given the central role of calcium in the regulation of signaling cascades and enzymatic activities within the cell. We also identified two proteins phosphorylated within putative PKC recognition motifs. PKC isoforms are involved in transducing signals received in the form of second messengers such as diacylglycerol, calcium, and inositol triphosphate, which result from activation of receptor tyrosine kinases and G protein-coupled receptors by extracellular ligands. Because the changing extracellular environment is expected to influence cellular metabolic needs, one might predict that the adiposome is subject to regulation by PKC. Phosphorylation was identified at one site corresponding to the recognition sequence of Akt, a protein kinase implicated as a mediator of extracellular and intracellular signaling events. The effects of Akt range from inhibition of apoptosis to transduction of insulin signaling cascades and regulation of the cell cycle. As an energy storage

technical notes

Kim et al.

Table 1. Adiposomal Phosphoproteinsa protein name

GI no.

peptide sequence

Signal Transduction 16933567 LEGNSPQG pSNQGVK 16933567 LEGN pSPQG pSNQGVK 337358 EDEI pSPPPPNPVVK 23943920 HIVSNDSSD pSDDESHEPK 3005717 RVQ pSLPSVPLSCAAYR 3005717 NNLpSLGDALAK 54695838 ADLA pSK 5453916 LLKPGEEPSEY pTDEEDTK 422774 AEEDEILNR pSPR 422774 QK pSDAEEDGGTV pSQEEEDRKPK Transcription/Translation centrosomal protein 1 34535303 KI pSEAGK ribosomal protein P1 isoform 1 4506669 KEESEE pSDDDMGFGLFD unnamed protein product (hypothetical protein FLJ10005) 10437063 DGQDAIAQ pSPEKESK DNA topoisomerase (ATP-hydrolyzing) 105857 IKNENTEG pSPQEDGVELEGLK Protein Transport vacuolar sorting protein 4 5381417 G pSDSDSEGDNPEKK sortilin precursor 84028263 SGYHDD pSDEDLLE translocation protein 1 4507525 SDSEEK pSDSEK Structural myosin, heavy polypeptide 9, non-muscle 34526505 KGAGDG pSDEEVDGKADGAEA KPAE erythrocyte membrane protein band 4.1-like 1 2224617 SEAEEGEVR pTPTK stromal interaction molecule 1 precursor 17368447 AEQ pSLHDLQER Lamin Adel10 14290259 LRL pSPSPTSQR Chaperone Hsp70-interacting protein 4928064 LGAGGG pSPEKSPSAQELK HSPCB protein 33987931 IEDVG pSDEEDDSGKDKK heat shock 90kDa protein 1, alpha 32486 DKEV pSDDEAEEK novel DnaJ domain protein 11125675 SL pSTSGESLYHVLGLDK Metabolism NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa 7770127 VAV pTPPGLAR acetyl-CoA carboxylase 1 33112873 FIIGSVSEDN pSEDEISNLVK acetyl-CoA carboxylase 1 33112873 SSM pSGLHLVK ubiquitin specific protease 33 5689531 LSA pSPPK ancient ubiquitous protein 1 isoform 1 6912260 LRPQSAQSSFPPSPGP pSPDVQLATLAQR Unknown hypothetical protein 12053153 DPLLFKSASD pTNLQK tumor differentially expressed 2 11282574 pSDGSLEDGDDVHR Mel transforming oncogene Mel transforming oncogene RET tyrosine kinase (cAMP dependent) phospholipase A2, group IVA patatin-like phospholipase domain containing 2 patatin-like phospholipase domain containing 2 RAB5C, member RAS oncogene family progesterone membrane binding protein calnexin calnexin

Mascot score

45 36 48 70 66 38 32 33 37 41 28 45 31 60 31 29 60 96 37 45 39 43 66 48 56 37 86 57 38 46 29 36

a Adiposomes from HeLa Cells were isolated, and proteins were extracted with 8 M urea, precipitated with TCA/acetone, and resuspended in 100 mM NH4HCO3. The suspension was digested with trypsin and subjected to IMAC as described in Materials and Methods, and the resulting peptides were analyzed by nano-HPLC/mass spectrometry. Identified phosphoproteins are grouped according to their functional annotation. For peptide sequences, a ‘p’ in front of S Or T indicates the phosphorylation site.

depot and active metabolic organelle, one might expect the adiposome to be influenced by the action of Akt. The discovery of such a contingent of adiposomal phosphoproteins is novel and suggests that the functions of the organelle are actively regulated by phosphorylation events. Furthermore, the results demonstrate that the clean digestion procedure is amenable to downstream sample enrichment strategies without the need for desalting or buffer exchange.

Discussion We developed a clean method for in-solution digestion of a protein mixture. The resulting tryptic peptides can be dried in SpeedVac to remove solvents and buffer, producing tryptic peptides devoid of detergents, urea, and salts. The results from exhaustive identification of peptides from A431 whole-cell lysate suggest that the protocol produces almost completely digested tryptic peptides (∼96.8% peptides with 0 missed cleavage sites), and more complete digestion than obtained in the presence of dilute (1 M) urea (87.3% peptides with 0 missed cleavage sites). It is likely that the clean digestion method described here will be applicable to proteolytic digestion using other enzymes. Using the clean digestion method eliminates the need for additional steps to remove salts, urea, or detergents

before HPLC/MS/MS analysis, or before methylating carboxylic acids prior to IMAC. The described digestion method is highly amenable to phosphoproteomic analysis. Previously, digestion of cell lysates has almost exclusively been performed in 1-2 M urea. Phosphopeptides are lost with this method because of the need to desalt tryptic peptides before methylation. When peptides are produced by our digestion procedure, desalting is necessary only after methylation and IMAC, resulting in improved recovery of phosphopeptides because methylated phosphopeptides have higher hydrophobicity. The absence of agents such as urea also facilitates complete removal of water, an important factor for complete methylation. Thus, the improved digestion procedure should be valuable due to improved recovery of phosphopeptides and more complete methylation of carboxylic acids. The advantages of the method are illustrated in the phosphoproteomic analysis of HeLa cell adiposomes, in which 33 phosphorylation sites were identified among 32 peptides from 28 proteins.

Acknowledgment. Y.Z. is supported by The Robert A. Welch Foundation (I-1550) and NIH (CA 107943). Journal of Proteome Research • Vol. 5, No. 12, 2006 3451

technical notes

Clean In-Solution Digestion of Protein Mixtures

References (1) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422, 198-207. (2) Washburn, M. P.; Wolters, D.; Yates, J. R. 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001, 19, 242-247. (3) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. 3rd. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 1999, 17, 676-682. (4) Park, Z. Y.; Russell, D. H. Thermal denaturation: A useful technique in peptide mass mapping. Anal. Chem. 2000, 72, 26672670. (5) Park, Z. Y.; Russell, D. H. Identification of individual proteins in complex protein mixtures by high-resolution, high-mass-accuracy MALDI TOF-mass spectrometry analysis of in-solution thermal denaturation/enzymatic digestion. Anal. Chem. 2001, 73, 25582564. (6) Russell, W. K.; Park, Z. Y.; Russell, D. H. Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry. Anal. Chem. 2001, 73, 26822685. (7) Pramanik, B. N. et al. Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: a new approach to protein digestion in minutes. Protein Sci. 2002, 11, 2676-2687. (8) Quach, T. T. T. et al. Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins. J. Proteome. Res. 2003, 2, 543552. (9) Zhang, N.; Li, L. Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass

3452

Journal of Proteome Research • Vol. 5, No. 12, 2006

(10)

(11)

(12)

(13)

(14)

(15)

spectrometric analysis of the digested peptides using two-layer sample preparation. Rapid Commun. Mass Spectrom. 2004, 18, 889-896. Li, N.; Shaw, A. R. E.; Zhang, N.; Mak, A.; Li, L. Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatographymatrix-assisted laser desorption/ionization tandem mass spectrometry. Proteomics 2004, 4, 3156-3166. Yu, Y. Q.; Gilar, M.; Gebler, J. C. A complete peptide mapping of membrane proteins: a novel surfactant aiding the enzymatic digestion of bacteriorhodopsin. Rapid Commun. Mass Spectrom. 2004, 18, 711-715. Liu, P.; Ying, Y.; Zhao, Y.; Mundy, D. I.; Zhu, M.; Anderson, R. G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004, 279, 3787-3792. Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20, 301-305. Chen, Y.; Kwon, S. W.; Kim, S. C.; Zhao, Y. Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra. J. Proteome Res. 2005, 4, 998-1005. Shannon, J. Using proteinases for Edman sequence analysis and peptide mapping. In Proteolytic Enzymes: A Practical Approach; Beynon, R., Bond, J. S., Eds., Oxford University Press: New York, NY, 2001; pp 187-210.

PR0603396