Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests

Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.     

Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry

A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

Proteome profiling of human cerebrospinal fluid: exploring the potential of capillary electrophoresis with surface modified capillaries for analysis of complex biological samples

A bottom-up proteomic approach, based on capillary electrophoresis (CE) in combination with matrix- assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToF MS), was used to analyze immunoaffinity depleted human cerebrospinal fluid (CSF) and compare it with a non-depleted sample. After enzymatic digestion and desalting, the tryptic peptides were separated by CE using PolyE-323 modified capillaries and fractionated off-line onto MALDI target plates for further analysis by MALDI-MS and MS/MS. The protein profile of the depleted sample was compared with non depleted CSF. Overall, 85 proteins were identified with 95% significance in both samples. The significance scores for proposed biomarkers, such as amyloid-like protein 1 precursor, could be increased up to 12 times after the depletion. Other proteins, often suggested to be related to neurodegenerative diseases, like amyloid beta A4 protein precursor, superoxide dismutase and apolipoprotein E precursor could only be found in the depleted CSF samples. The effect of a derivatization of tryptic peptides with 2- methoxy-4,5-dihydro-1H-imidazole reagent for protein identification with MS was also employed to increase the number of identified proteins and the sequence coverages. The results presented in this study illustrate the benefit of combining a sample pre-fractionation step and a label's ability to enhance the ionization efficiency with the potential of CE using PolyE-323 modified capillaries in the analysis of complex samples. The straight-forward approach that provides speed and simplicity resulting in high-resolution separations and low sample consumption represents an easily applicable separation technique that can serve as a complement to other currently existing analytical approaches needed in modern proteomic analysis of clinically relevant samples.     

Nanodiamond MALDI support for enhancing the credibility of identifying proteins

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a standard analytical tool for protein identification and peptide sequencing. High sensitivity and resolution are two critical parameters for recording good peptide mass fingerprinting (PMF) of low abundance proteins. Here, we report a novel nanodiamond (ND) (normal size 3–10 nm) support for MALDI-MS target, over which -cyano-4-hydrocinnamic acid (CCA) crystallizes evenly. Good reproducibility of relative peak intensity (R.S.D. less than 11.8%) among sample spot (from ring to center) is achieved on ND support. Therefore, the search for “hot spots” during the analysis is not necessary, which is supporting for the automatic acquisition of data. Due to high absorbability of energy from the laser, the ND support improves ionization efficiency of samples. In general, the sensitivity of MS obtained on ND support can be enhanced three to four times compared to the conventional MALDI sample preparation technique. Sensitivity obtained on ND support ranges from 62.5 amol of Arg-vasopressin standard peptide to 1.0 fmol of myoglobin tryptic peptide mixture. Reduced spot size and increased sensitivity in MALDI-MS are also accomplished by ND support. With spot size reduced, the signal intensity of cytochrome c (Cyt c) tryptic peptide obtained on ND support is at least seven times greater than it acquired on stainless steel. And ND support has been found better tolerance for salt (up to 500 mM NaCl) to MALDI-MS analysis. All these properties make ND support a valuable tool for MALDI-MS identification of proteins.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry

Mass spectrometry was applied to identify protein spots excised from an archived two-dimensional polyacrylamide gel that had been dried and stored for eight years at room temperature. All proteins were successfully identified. Detailed characterization of protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mapping, nanoelectrospray tandem mass spectrometry and MALDI-quadrupole time-of-flight mass spectrometry revealed no evidence of protein degradation or modifications that could hamper identification of proteins in a sequence database. The experiment with a model protein demonstrated that the pattern of tryptic peptides and the yield of individual peptides were not noticeably changed in the in-gel digest of the archived protein spot compared to the digest of the spot excised from a fresh gel. Thus, the characterization of "archived proteomes" has the potential to advance proteomic research without repeating "wet" biochemistry experiments, that had been perfected in the laboratory years ago.     

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization--mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.     

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

Preparation and characterization of ceria‐zirconia composite for enrichment and identification of phosphopeptides

Ceria‐zirconia composites at different molar ratios were synthesized. Several methods were used to characterize these composites, including X‐ray photoelectron spectroscopy, surface area and surface acid‐base property detection. A one‐step method for isolation and identification of phosphopeptides from peptide mixture was created using these ceria‐zirconia composites. Using tryptic digest of standard phosphorylated protein, we have shown that these enrichment and dephosphorylation activities are effective. The adsorption capacity and catalytic property of ceria‐zirconia composites at different molar ratios and calcinated temperatures were studied. In combination with MALDI‐TOF‐based peptide mass finger printing technique, we have established a method to utilize the enrichment/dephosphorylation dual properties of these ceria‐zirconia composites for the analysis of phosphoprotein in nonfat milk successfully.     

Proteome analysis of a human heptocellular carcinoma cell line, HCC-M: an update.

Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC-M (Electrophoresis 2000, 21, 1787-1813), using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). From a total of 408 unique spots excised from the 2-DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). First, "peptide tag sequencing" was performed to obtain partial amino acid sequences of the peptides to search the SWISS-PROTand NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novo peptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post-translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA-binding protein regulatory subunit, replication protein A 32 kDa subunit (RP-A) and N-acetylneuraminic acid phosphate synthase were reported to be cancer-related proteins.     

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.     

Signature-peptide approach to detecting proteins in complex mixtures

The objective of the work presented in this paper was to test the concept that tryptic peptides may be used as analytical surrogates of the protein from which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatography, lectin columns were used in this case. Affinity selected peptide mixtures were directly transferred to a high-resolution reversed-phase chromatography column and further resolved into fractions that were collected and subjected to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The presence of specific proteins was determined by identification of signature peptides in the mass spectra. Data are also presented that suggest proteins may be quantified as their signature peptides by using isotopically labeled internal standards. Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a peptide relative to that of the labeled internal standard. Peptides in tryptic digests were labeled by acetylation with acetyl N-hydroxysuccinimide while internal standard peptides were labeled with the trideuteroacetylated analogue. Advantages of this approach are that (i) it is easier to separate peptides than proteins, (ii) native structure of the protein does not have to be maintained during the analysis, (iii) structural variants do not interfere and (iv) putative proteins suggested from DNA databases can be recognized by using a signature peptide probe.     

Mass spectrometric study of the effects of hydrophobic surface chemistry and morphology on the digestion of surface-bound proteins

Our previous work has demonstrated that reversed-phase chromatographic micro-beads can be used to capture proteins from complex biological matrices and the surface-bound proteins can be enzymatically digested for protein identification by mass spectrometry (MS). Here we examine the peptides generated from digestion of proteins bound to various types of micro-bead surfaces in order to determine the effects of surface chemistry and surface morphology on the digestion process. Detailed examinations of site cleavages and sequence coverage are carried out for a tryptic digestion of cytochrome c adsorbed on reversed-phase polystyrene divinylbenzene (Poros R2 beads) versus C(18) bonded-phase silica beads. It is shown that although the surface does not completely hinder the digestion of cleavage sites of the protein, the digestion products are clearly different than those obtained from a solution digest. Specifically, a partial digestion results from surface digestion, resulting in a greater number of missed cleavages than a comparable solution digest. Subsequent comparisons of peptide mass maps generated from the digestion of various proteins on surfaces with altering chemistry (C(4), C(8), C(18), and R2 beads), or with different surface morphology, were performed. The results reveal that surface chemistry plays only a minor role in affecting the peptide mass maps, and surface morphology had no noticeable effects on the resulting peptide mass maps. It is also shown that the mass spectrometric detection method used to analyze the digested peptides can significantly influence the information content on cleavage sites and the extent of sequence coverage. The use of a combination of MALDI, LC/off-line MALDI, and LC/ESI MS is demonstrated to be crucial in revealing subtle changes in the peptide mass maps.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChip sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrate

The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).     

Effect of sodium dodecyl sulfate micelles on peptide mass fingerprinting by matrix-assisted laser desorption/ionization mass spectrometry

Here we have examined the effect of sodium dodecyl sulfate (SDS) at various concentrations on matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting experiments. Several model proteins were digested with trypsin and then various amounts of SDS were added prior to MALDI mass spectrometry. Evaluation of the data was made by calculating the amino acid sequence coverage within each analysis. It was found that addition of 0.1-0.3% w/v SDS prior to MALDI analysis results in an increase in the number of tryptic peptides detected thereby improving the total sequence coverage of the analysis. The use of SDS at concentrations near its critical micelle concentration can improve sequence coverage from MALDI peptide mass fingerprinting analyses allowing for increased confidence in protein identification or additional opportunities to identify putative regions of posttranslational modification.     

Ion soft-landing into liquids: Protein identification,separation, and purification with retention of biological activity

Protein ions, after mass spectrometric separation, can be soft-landed into liquid surfaces with preservation of their native structures. Retention of biological activity is strongly favored in glycerol-based surfaces but not in self-assembled monolayer solid surfaces. Soft-landing efficiency for multiply-charged hexokinase ions was found to be some four times higher for a glycerol/fructose liquid surface than for a fluorinated self-assembled monolayer surface. Soft-landing into liquid surfaces is also shown to allow (1) protein purification, (2) on-surface identification of the soft-landed material using MALDI, and (3) protein identification by in-surface tryptic digestion. Pure lysozyme was successfully isolated from different mixtures including an oxidized, partially decomposed batch of the protein and a partial tryptic digest. Liquid glycerol/carbohydrate mixtures could be used directly to record MALDI spectra on the soft-landed compounds provided they were fortified in advance with traditional MALDI matrices such as p-nitroaniline and alpha-cyano-4-hydroxycinnamic acid. Various proteins were soft-landed and detected on-target using these types of liquid surface. Soft-landing of multiply-charged lysozyme ions onto fluorinated self-assembled monolayer surfaces was found to occur with a limited amount of neutralization, and trapped multiply-charged ions could be desorbed from the surface by laser desorption. Initial data is shown for a new approach to protein identification that combines top-down and bottom-up approaches by utilizing protein ion soft-landing from a protein mixture, followed by tryptic digestion of the landed material and detection of characteristic tryptic fragments by MALDI.