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At least 19 recordsLinked to original sources

PubMed2005

Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection.

Despite major advances in mass spectrometry, the detection of phosphopeptides by liquid chromatography with electrospray mass spectrometry (LC/ES-MS) still remains very challenging in proteomics analysis. Phosphopeptides do not protonate efficiently due to the presence of one or more acidic phosphate groups, making their detection difficult. However, other mechanisms also contribute to the difficulties in phosphopeptide analysis by LC/ES-MS. We report here on one such undocumented problem: the formation of phosphopeptide-metal ion complexes during LC/ES-MS. It is demonstrated that both synthetic phosphopeptides and phosphopeptides from bovine beta-casein and alpha-casein form phosphopeptide-metal ion complexes containing iron and aluminum ions, resulting in a dramatic decrease in signal intensity of the protonated phosphopeptides. The interaction of phosphopeptides with metal ions on the surface of the C18 stationary phase is also shown to alter their chromatographic behavior on reversed-phase columns such that the phosphopeptides, especially multiply phosphorylated peptides, become strongly retained and very difficult to elute. The sources of iron and aluminum are from the solvents, stainless steel, glassware and C18 material. It was also found that, upon addition of EDTA, the formation of the phosphopeptide-metal ion complex is diminished, and the phosphopeptides that did not elute from the LC column can now be detected efficiently as protonated molecules. The sensitivity of detection was greatly increased such that a tetra-phosphorylated peptide, RELEELNVPGEIVEpSLpSpSpSEESITR from the tryptic digestion of bovine beta-casein, was detected at a limit of detection of 25 fmol, which is 400 times lower than without EDTA.

Animals↗

PubMed1999

Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues.

A set of synthetic phosphopeptides and phosphopeptide analogues was studied by tandem nano-electrospray mass spectrometry. The influence of the collision offset and of the charge state of the molecular ion on phosphate-specific fragmentation processes was investigated in detail. H--D exchange experiments and structural considerations support a six-centered transition being present in the neutral loss of H3PO4 from serine, threonine and homoserine phosphopeptides, where the C-alpha hydrogen of serine or threonine or the C-beta hydrogen of homoserine is transferred to the protonated phosphate group. Neutral loss of H3PO4 at moderate collision offset potential represents a very abundant fragmentation process for serine, threonine and homoserine phosphopeptides. The most specific feature for discrimination of these phosphopeptides from tyrosine phosphopeptides is the m/z 79:97 ratio in the negative ion product spectra, which is consistently elevated in tyrosine phosphopeptides as compared with serine, threonine and homoserine phosphopeptides. The fragment ions of methylphosphono- and H-phosphonopeptides can be explained by the same mechanisms as are applicable to phosphopeptides.

Deuterium↗

PubMed1993

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

Amino Acid Sequence↗

PubMed1996

Semi-preparative isolation of phosphopeptides derived from bovine casein and dephosphorylation of casein phosphopeptides.

Semi-preparative isolation of casein phosphopeptides (CPP) from a tryptic hydrolysate of bovine casein was performed applying a three-step procedure consisting of solid phase extraction, reversed phase HPLC and ion exchange chromatography. Dephosphorylation of CPP was achieved using immobilized alkaline phosphatase. The purified phosphopeptides and their dephosphorylated forms obtained by these methods are suitable for comparative studies on biological activities, especially mineral binding and immunmodulation.

Animals↗

PubMed1999

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics.

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees </= 0.6 kcal/mol) to conservative substitutions at all three peptide positions. However, mutation to Ala resulted in moderate reductions in DeltaG degrees, with the substitution at the +3 position showing the largest loss in affinity (DeltaDeltaG degrees = 1.4 kcal/mol), followed by the +2 (DeltaDeltaG degrees = 1.0 kcal/mol) and +1 (DeltaDeltaG degrees = 0.5 kcal/mol) positions. This hierarchy of binding was not reflected in the values of the heat capacity change, since only the peptide substituted to Ala at the +3 position showed a DeltaCp degrees that was reduced in magnitude compared to wild-type. To assess the degree of cooperation upon binding (or coupling) between the amino acids of the EEI sequence, the binding of a series of singly, doubly, and triply Ala substituted phosphopeptides was examined and analyzed using double mutant cycles. It was revealed that the effects of the Ala substitutions on DeltaG degrees were additive. However, nonadditive binding enthalpies were observed between the +1 Glu and +3 Ile, as well as the +2 Glu and +3 Ile, suggesting that communication occurs between residues of the EEI motif upon binding.

Alanine↗

PubMed2005

Identification of phosphopeptides by MALDI Q-TOF MS in positive and negative ion modes after methyl esterification.

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.

Caseins↗

PubMed2006

Enrichment of phosphopeptides by Fe3+-immobilized mesoporous nanoparticles of MCM-41 for MALDI and nano-LC-MS/MS analysis.

Fe3+-immobilized mesoporous molecular sieves MCM-41 with particle size of ca. 600 nm and pore size of ca. 3 nm is synthesized and applied to selectively trap and separate phosphopeptides from tryptic digest of proteins. For the capture of phosphopeptides, typically 10 microL of tryptic digest solution was first diluted to 1 mL by solution of ACN/0.1% TFA (50:50, v/v) and incubated with 10 microL of 0.1% acetic acid dispersed Fe3+-immobilized MCM-41 for 1 h under vibration. Fe3+-immobilized MCM-41 with trapped phosphopeptides was separated by centrifugation. The deposition was first washed with a volume of 300 microL of solution containing 100 mM NaCl in ACN/0.1% TFA (50:50, v/v) and followed by a volume of 300 microL of solution of 0.1% acetic acid to remove nonspecifically bound peptides. The nanoparticles with trapped phosphopeptides are mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and deposited onto the target for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that phosphopeptides from tryptic digest of alpha-casein and beta-casein are effectively and specifically trapped on Fe3+-immobilized MCM-41 with few peptides nonspecifically adsorbed. After the extraction by Fe3+-immobilized MCM-41, the suppression to the detection of phosphopeptides caused by abundant nonphosphopeptides from tryptic digest is effectively eliminated, and the detection of phosphopeptides by MALDI is greatly enhanced with the value of signal-to-noise (S/N) increased by more than an order of magnitude. It is demonstrated that the mechanism of the adsorption of phosphopeptides on Fe3+-immobilized MCM-41 is based on the interaction between the Fe3+ and the phosphate group. Finally, Fe3+-immobilized MCM-41 is applied to extract phosphopeptides from tryptic digest of the lysate of mouse liver for phosphoproteome analysis by nano-LC-MS/MS.

Amino Acid Sequence↗

PubMed2006

Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis.

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.

Amino Acid Sequence↗

PubMed1999

Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry.

The analysis of large phosphoproteins by mass spectrometry is a particular challenge, in many cases, because of the small proportion of phosphopeptides in the presence of a large number of non-phosphorylated peptides. In addition, phosphopeptides are generally available in dilute solutions. Thus, methods to specifically identify phosphopeptides at low concentrations are important. In this work, on-line Fe(III) immobilized metal-ion affinity chromatography (IMAC)-CE-electrospray ionization MS was developed and applied to sub-pmol analysis of phosphopeptides. Phosphopeptides bind Fe(III) with high selectivity. The IMAC resin is packed directly at the head of the CE column. After the phosphopeptides are bonded to the resin and washed, they are eluted at high pH and separated by CE. This method has several advantages: (1) selective retention and pre-concentration of phosphopeptides on an Fe(III)-IMAC resin; (2) a pre-wash of the sample to remove salts and buffers that are not suited for CE separation or ESI operation; (3) facile fabrication with common tools and chemicals (less than 10 min); (4) adaptation to commercial CE instruments without any modifications. The applications of IMAC-CE-MS are demonstrated by the analysis of phosphopeptide mixtures and a phosphoprotein digest.

Amino Acid Sequence↗

PubMed2005

Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis.

We have developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The method employs a biphasic IMAC-C18 tip, in which IMAC beads are packed on an Empore C18 disk in a 200-microL pipet tip. Phosphopeptides are separated from non-phosphopeptides on the IMAC in an optimized solvent without any chemical reaction, then desorbed from the IMAC using a phosphate buffer, reconcentrated, and desalted on the C18 disk. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides and (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides. The entire procedure was optimized by using known phosphoproteins such as Akt1 kinase and protein kinase A. Although it was difficult to detect phosphopeptides in MALDI-MS spectra of tryptic peptide mixtures before enrichment, after the IMAC procedure, we could successfully detect phosphopeptides with almost no non-phosphopeptides. Next, we constructed an array of IMAC-IMAC/C18 tips, such that number of arrayed tips on a 96-well plate could easily be changed depending on the loading amount of sample. Applying this approach to mouse forebrain resulted in the identification of 162 phosphopeptides (166 phosphorylation sites) from 135 proteins using nano-LC/MS.

Amino Acid Sequence↗

PubMed2006

Fast track to a phosphoprotein sketch - MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity.

Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.

Chromatography, Thin Layer↗

PubMed1993

Phosphopeptides as substrates for thylakoid protein phosphatase activity.

Lack of a suitable substrate has been a major obstacle in studying the chloroplastic thylakoid membrane protein phosphatase activity. In this study, the suitability of synthetic phosphopeptides for this purpose was investigated. Phosphothreonine-containing phosphopeptides mimicking the N-terminal phosphorylation site of the major thylakoid phosphoprotein, the light-harvesting chlorophyll a/b-binding protein (LHCP-II), were dephosphorylated by isolated peak thylakoid membranes. Phosphopeptides representing unrelated sequences or in which the target phosphothreonine had been changed to a phosphoserine were not dephosphorylated. The dephosphorylation of phosphopeptides by thylakoid membranes was similar to the dephosphorylation of endogenous LHCP-II in its pH-dependence profile, sensitivity to inhibitors, and bivalent cation requirement. The same phosphopeptide analogs of the LHCP-II phosphorylation site inhibited endogenous LHCP-II dephosphorylation in isolated thylakoids, whereas the dephospho-analogs and nonsubstrate phosphopeptides had no effect. Collectively, these results suggest that phosphopeptides mimicking a thylakoid phosphoprotein dephosphorylation site can be exploited for further study of the thylakoid protein phosphatase activity.

Amino Acid Sequence↗

PubMed1994

Evaluation of two-dimensional phosphopeptide maps by electrospray ionization mass spectrometry of recovered peptides.

Intracellular signaling pathways are to a large extent regulated by reversible protein phosphorylation of pathway components. To fully investigate the regulation of these pathways, it is often necessary to identify the sites of protein phosphorylation induced on individual components. The low abundance of many of these molecules and the potentially low stoichiometry of phosphorylation means that conventional analytical techniques are incapable of identifying specific sites of modification inducible in vivo. The most common technique used is two-dimensional (2D) phosphopeptide mapping (electrophoresis, thin-layer chromatography) of peptides derived by proteolysis of a phosphoprotein. The number of spots detected is commonly interpreted as the number of sites of phosphorylation. Here we have achieved positive identification of phosphorylation sites by capillary high-performance liquid chromatography, with on-line mass spectrometric detection, of phosphopeptides recovered from 2D phosphopeptide maps. We demonstrate that the chemical composition of phosphopeptides is not altered during the 2D mapping procedure. By detailed analysis of the sites of phosphorylation induced in vitro on CD3-zeta by p56lck we demonstrate that interpretation of the sites of phosphorylation based on 2D phosphopeptide mapping alone is difficult. To minimize over- or misinterpretation of 2D phosphopeptide maps we therefore postulate rules that should be applied generally in cases in which protein phosphorylation sites are being evaluated by 2D phosphopeptide patterns alone.

Amino Acid Sequence↗

PubMed1996

Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry.

We describe a new procedure that enables selective detection and sequencing of Ser-, Thr-, and Tyr-phosphopeptides at the low femtomole level in protein digests. Radiolabeling with 32P is not required, nor is prior chromatographic separation of the peptide mixture. One to two microliters of the unfractionated protein digest is infused at basic pH into an electrospray mass spectrometer at a flow rate of 20-40 nl/min using an ultra-low flow sprayer. A precursor-ion scan of m/z 79 (PO-3) produces a mass spectrum containing only the molecular ions of the phosphopeptides that are present in the sample. In cases where the protein sequence is known, the peptide molecular weights obtained are often sufficient to identify the specific sequences that are phosphorylated. If the protein sequence is not known, tandem MS with collision-induced dissociation of phosphopeptide precursor-ions may be used to obtain the amino acid sequences including the site(s) of phosphorylation. We demonstrate that phosphopeptides may be selectively detected using as little as 3 fmol of a 10 fmol/microl solution and that sequence information for a phosphopeptide in the mixture may be obtained using as little as 3 femtomole of the same solution. In addition, we show that the stoichiometry of phosphorylation at specific sites may be estimated from the ratio of the ion signals for the respective forms of the peptides observed in the normal full-scan mass spectra of the digest. These procedures are illustrated here to identify and sequence phosphopeptides from alpha-casein, a milk-derived protein possessing up to nine phosphorylation-sites. Numerous MS and tandem MS experiments were carried out on a single, 250 fmol/microl loading of the phosphoprotein digest. Phosphopeptides derived from an unexpected variant of the protein were also observed.

Amino Acid Sequence↗

PubMed2001

Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.

Animals↗

PubMed2004

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).

Amino Acid Sequence↗

PubMed2005

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry.

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.

Amino Acid Sequence↗

PubMed1976

Identification and subcellular distribution of adipocyte peptides and phosphopeptides.

Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.

Adipose Tissue↗