Tuning of an electrospray ionization source for maximum peptide-ion transmission into a mass spectrometer.

We describe assembly and optimization of a continuous flow nanoelectrospray source for high-performance analysis on a routine basis. It is derived from an inJection adaptable Fine Ionization Source ("JaFIS"), previously shown to be durable and easy to use (Geromanos, S.; et al. Rapid Commun. Mass Spectrom. 1998, 12, 551-556) and now modified for maximum sensitivity. Proper design, manufacturing, and quality control of spray needles with specific orifice diameters, in combination with precisely controlled helium backpressure and applied voltage, enable stable flows at 1-2 nL/min. Needle positioning and ion spray potential are hereby exceedingly important, as shifts by 0.5 mm or 25 V, respectively, cause significant reduction in signal strength. In addition to prolonged analysis times, ultralow flows also yield higher sensitivity, the result of an improved "overall ion transfer efficiency" measured to be approximately 5% at 1.6 nL/min. Used in combination with a "microtip" (Erdjument-Bromage, H.; et al. J. Chromatogr. A 1998, 826, 167-181), the optimized JaFIS implements infusion-style ESI-MS at sensitivities approaching capillary LC-MS. Spraying times in excess of 20 h allow for any number of tandem mass spectrometric analysis routines to be performed, and to average thousands of scans in every experiment, thereby further improving sensitivity. This was fully illustrated by extensive analysis of a 2-fmol peptide mixture, in a 2-microL volume, using a multimode MS approach.

A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells.

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.

InJection adaptable fine ionization source ('JaFIS') for continuous flow nano-electrospray.

Nanoelectrospray ('nanoES') tandem mass spectrometry of complex peptide mixtures has become a certified, mostly reliable technique for the identification of proteins. The typical low flow rates of nanoES, the extended analysis times for small samples, high ion transmission and its overall ease-of-use provide important practical advantages for polypeptide covalent microanalysis. We have constructed a modified nanoES ion source that is highly durable and user-friendly, and potentially allows for full auto-sampling operation. The inJection adaptable Fine Ionization Source ('JaFIS') can be operated at flow rates of 10-100 nL per minute and with sensitivities in the 25 femtomoles peptide per microliter range. The ion spray needles usually last for days, allowing for standards and multiple samples to be analyzed consecutively under similar conditions. In this configuration, quality controlled needles can also be saved and reused, providing for more consistent and reproducible day-to-day operating conditions, JaFIS-ES also permits sample recovery should any failure occur during analysis.

The propeptide binding site of the bovine gamma-glutamyl carboxylase.

gamma-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the gamma-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the gamma-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine gamma-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles.

Rab proteins are generally required for transport vesicle docking. We have exploited yeast secretion mutants to demonstrate that a rab protein is required for v-SNAREs and t-SNAREs to assemble. The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly. In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking. This mechanism likely involves the Sec1 homolog Sly1, which we identified in isolated docking complexes. We also report the identification of a novel v-SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has a CAAX box and is predicted to be lipid anchored. The surprising finding that docking complexes can contain many distinct species of SNAREs (Sed5p, Bos1p, Sec22p, Ykt6p, and likely Bet1p, p28, and p14) suggests that multimeric interactions are features of the fusion machinery, and may also improve the fidelity of vesicle targeting.

Microbore reversed-phase high-performance liquid chromatographic purification of peptides for combined chemical sequencing-laser-desorption mass spectrometric analysis.

An optimized microbore RP-HPLC system (1.0 mm I.D. columns) for the purification of low picomole amounts (< 5 pmol) of peptides is described. It is comprised of commercially available columns, instrument components and parts. These were selected on the basis of a comparative evaluation and to yield the highest resolution and most efficient peak collection. The sensitivity of this system equals, probably surpasses, that of advanced chemical microsequencing for which 2-4 pmol of peptide are minimally required. As an automated sequencer cannot be "on-line" connected with a micro-preparative HPLC system, fractions must be collected and transferred. With a typical flow of 30 microliters, efficient manual collection is possible and fractions (about 20 microliters in volume) can still be handled without unacceptable losses, albeit with great precaution. Furthermore, major difficulties were encountered to efficiently and quantitatively load low- or sub-picomole amounts of peptide mixtures onto the RP-HPLC column for separation. Discipline and rigorous adherence to sample handling protocols are thus on order when working at those levels of sensitivity. With adequate instrumentation and handling procedures in place, we demonstrate that low picomole amounts of peptides can now be routinely prepared for analysis by combined Edman-chemical sequencing-matrix-assisted laser-desorption mass spectrometry (MALDI-MS). The integrated method was applied to covalent structural characterization of minute quantities of a gel-purified protein of known biological function but unknown identity. The results allowed unambiguous identification and illustrated the power of MALDI-MS-aided interpretation of chemical sequencing data: accurate peptide masses were crucial for (i) confirmation of the results, (ii) deconvolution of mixed sequences, (iii) proposal of complete structures on the basis of partial sequences, and (iv) confirmation of protein identification (obtained by database search with a single, small stretch of peptide sequence) by "mass matching" of several more peptides with predicted proteolytic fragments.

Identification of nucleotide-binding regions in the chaperonin proteins GroEL and GroES.

The chaperonin GroEL, a tetradecameric cylinder consisting of subunits of M(r) approximately 60,000 (60K), and its cofactor GroES, a heptameric ring of 10K subunits, mediate protein folding in the cytosol of Escherichia coli. In the presence of nucleotide, GroES forms a 1:1 complex with GroEL which binds unfolded protein in its central cavity and releases it to allow folding upon ATP hydrolysis. Using labelling with azido-ATP, we have identified a protease-stable nucleotide-binding domain of M(r) 40K in the GroEL subunits (residues 153-531). Azido-ATP is crosslinked to the highly conserved Tyr 477, indicating that this residue is close to the purine ring of the bound nucleotide. Surprisingly, GroES also binds ATP cooperatively and with an affinity comparable to that of GroEL. Azido-nucleotide labelling of GroES subunits occurs at the conserved Tyr 71 in a protease-stable 6.5K domain (starting at residue 33). Proteinase K cleavage at residue 32 is prevented when GroES is bound to GroEL. ATP binding to GroES may be important in charging the seven subunits of the interacting GroEL ring with ATP to facilitate cooperative ATP binding and hydrolysis for substrate protein release.

SNAP receptors implicated in vesicle targeting and fusion.

The N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs) appear to be essential components of the intracellular membrane fusion apparatus. An affinity purification procedure based on the natural binding of these proteins to their targets was used to isolate SNAP receptors (SNAREs) from bovine brain. Remarkably, the four principal proteins isolated were all proteins associated with the synapse, with one type located in the synaptic vesicle and another in the plasma membrane, suggesting a simple mechanism for vesicle docking. The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicle or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesicle-to-target specificity.